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Journal of Structural Biology
Journal Prestige (SJR): 3.948
Citation Impact (citeScore): 3
Number of Followers: 5  
 
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 1047-8477 - ISSN (Online) 1095-8657
Published by Elsevier Homepage  [3157 journals]
  • 3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the
           recruitment of host cell organelles towards parasitophorous vacuoles
           during its biogenesis
    • Abstract: Publication date: Available online 17 January 2019Source: Journal of Structural BiologyAuthor(s): Lissa Catherine Reignault, Carolina de Lima Alcantara, Emile Santos Barrias, Wanderley de Souza Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.
       
  • NADPH-dependent sulfite reductase flavoprotein adopts an extended
           conformation unique to this diflavin reductase
    • Abstract: Publication date: Available online 15 January 2019Source: Journal of Structural BiologyAuthor(s): Angela M. Tavolieri, Daniel T. Murray, Isabel Askenasy, Joseph M. Pennington, Lauren McGarry, Christopher B. Stanley, M. Elizabeth Stroupe This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.Graphical abstractGraphical abstract for this article
       
  • Response of the tooth-periodontal ligament-bone complex to load: a microCT
           study of the minipig molar
    • Abstract: Publication date: Available online 11 January 2019Source: Journal of Structural BiologyAuthor(s): Yehonatan Ben-Zvi, Raquel Maria, Maria Pierantoni, Vlad Brumfeld, Ron Shahar, Steve Weiner One strategy evolved by teeth to avoid irreversible damage is to move and deform under the loads incurred during mastication. A key component in this regard is the periodontal ligament (PDL). The role of the bone underlying the PDL is less well defined. We study the interplay between the PDL and the underlying alveolar bone when loaded in the minipig. Using an Instron loading device we confirmed that the force-displacement curves of the molars and premolars of relatively fresh minipig intact mandibles are similar to those obtained for humans and other animals. We then used this information to obtain 3D images of the teeth before and after loading the tooth in a microCT such that the load applied is in the third linear part of the force displacement curve. We observed that at many locations there is a complimentary topography of the cementum and alveolar bone surface, strongly suggesting an active interplay between the tooth and the bone during mastication. We also observed that the loaded tooth does not come into direct contact with the underlying bone surface. A highly compressed layer of PDL is present between the tooth and the bone. The structure of the bone in the upper furcation region has a unique appearance with little obvious microstructure, abundant pores that have a large size range and at many locations the bone at the PDL interface has a needle-like shape. We conclude that there is a close interaction between the tooth, the PDL and the underlying alveolar bone during mastication. The highly compressed PDL layer that separates the tooth from the bone may fulfill a key shock absorbing function.Graphical abstractGraphical abstract for this article
       
  • Rapid Tilt-Series Acquisition for Electron Cryotomography
    • Abstract: Publication date: Available online 11 January 2019Source: Journal of Structural BiologyAuthor(s): Georges Chreifi, Songye Chen, Lauren Ann Metskas, Mohammed Kaplan, Grant J. Jensen Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ∼4 nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.Graphical abstractGraphical abstract for this article
       
  • Using neutron crystallography to elucidate the basis of selective
           inhibition of carbonic anhydrase by saccharin and a derivative
    • Abstract: Publication date: Available online 11 January 2019Source: Journal of Structural BiologyAuthor(s): Katarina Koruza, Brian P. Mahon, Matthew P. Blakeley, Andreas Ostermann, Tobias E. Schrader, Robert McKenna, Wolfgang Knecht, S. Zoë Fisher Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.Graphical abstractJoint X-ray and neutron crystallographic studies reveal details of carbonic anhydrase (CA) isoform inhibitor selectivity. Only neutron studies can provide details of water and H-bond reorganization, and the involvement of specific CA IX amino acid residues interacting with the inhibitors. This approach can provide clues to future CA isoform selective inhibitor design.Graphical abstract for this article
       
  • The FKBP12 subunit modifies the long-range allosterism of the ryanodine
           receptor
    • Abstract: Publication date: Available online 11 January 2019Source: Journal of Structural BiologyAuthor(s): Tyler W.E. Steele, Montserrat Samsó Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR’s constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca2+. FKBP12 binds to RyR1’s cytoplasmic assembly 130 Å away from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1’s channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1’s conformational landscape.Graphical abstractGraphical abstract for this article
       
  • Structural transformation-mediated dimerization of caspase recruitment
           domain revealed by the crystal structure of CARD-only protein in frog
           virus 3
    • Abstract: Publication date: Available online 6 January 2019Source: Journal of Structural BiologyAuthor(s): Chang Min Kim, Hyun Ji Ha, Sunghark Kwon, Jae-Hee Jeong, Sung Hoon Lee, Yeon-Gil Kim, Chang Sup Lee, Jun Hyuck Lee, Hyun Ho Park Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.Graphical abstractGraphical abstract for this article
       
  • Structural and functional characterization of TgpA, a critical protein for
           the viability of Pseudomonas aeruginosa
    • Abstract: Publication date: Available online 30 December 2018Source: Journal of Structural BiologyAuthor(s): Mónica Uruburu, Eloise Mastrangelo, Martino Bolognesi, Silvia Ferrara, Giovanni Bertoni, Mario Milani Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6 Å resolution as a first step towards structure-activity analysis of a new potential target for the discovery of antibacterial compounds.
       
  • Effects of the cardiomyopathy-causing E244D mutation of troponin T on the
           structures of cardiac thin filaments studied by small-angle X-ray
           scattering
    • Abstract: Publication date: Available online 30 December 2018Source: Journal of Structural BiologyAuthor(s): Tatsuhito Matsuo, Fumiaki Kono, Satoru Fujiwara Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.Graphical abstractGraphical abstract for this article
       
  • Bending and branching of calcite laths in the foliated microstructure of
           pectinoidean bivalves occurs at coherent crystal lattice orientation
    • Abstract: Publication date: Available online 19 December 2018Source: Journal of Structural BiologyAuthor(s): Antonio G. Checa, María E. Yáñez-Ávila, Alicia González-Segura, Francisco Varela-Feria, Erika Griesshaber, Wolfgang W. Schmahl Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.Graphical abstractGraphical abstract for this article
       
  • Structural and functional characterization of a family GH53
           β-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates
           degradation of prebiotic galactooligosaccharides
    • Abstract: Publication date: Available online 13 December 2018Source: Journal of Structural BiologyAuthor(s): Markus Böger, Johan Hekelaar, Sander S. van Leeuwen, Lubbert Dijkhuizen, Alicia Lammerts van Bueren Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-Ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤ 3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.Graphical abstractGraphical abstract for this article
       
  • Structural insights into thermostabilization of leucine dehydrogenase from
           its atomic structure by cryo-electron microscopy
    • Abstract: Publication date: Available online 10 December 2018Source: Journal of Structural BiologyAuthor(s): Hiroki Yamaguchi, Akiko Kamegawa, Kunio Nakata, Tatsuki Kashiwagi, Toshimi Mizukoshi, Yoshinori Fujiyoshi, Kazutoshi Tani Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain L-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2 Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80°C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.Graphical abstractGraphical abstract for this article
       
  • A Local Agreement Filtering Algorithm for Transmission EM Reconstructions
    • Abstract: Publication date: Available online 29 November 2018Source: Journal of Structural BiologyAuthor(s): Kailash Ramlaul, Colin M Palmer, Christopher H S Aylett We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM.Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps.LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 2563-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available.We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) Cref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.Graphical abstractGraphical abstract for this article
       
  • Metal centers in biomolecular solid-state NMR
    • Abstract: Publication date: Available online 28 November 2018Source: Journal of Structural BiologyAuthor(s): José Malanho Silva, Linda Cerofolini, Stefano Giuntini, Vito Calderone, Carlos F.G.C. Geraldes, Anjos L. Macedo, Giacomo Parigi, Marco Fragai, Enrico Ravera, Claudio Luchinat Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.
       
  • goCTF: Geometrically optimized CTF determination for single-particle
           cryo-EM
    • Abstract: Publication date: Available online 26 November 2018Source: Journal of Structural BiologyAuthor(s): Min Su Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 Å to 3.7 Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.Graphical abstractGraphical abstract for this article
       
  • Pig enamel revisited - Incremental markings in enamel of wild boars and
           domestic pigs
    • Abstract: Publication date: Available online 22 November 2018Source: Journal of Structural BiologyAuthor(s): Horst Kierdorf, Friederike Breuer, Carsten Witzel, Uwe Kierdorf The nature and periodicity of incremental markings in pig enamel is currently debated. To broaden the basis for a correct interpretation of growth marks in pig enamel, we analyzed their periodicity in teeth of wild boars and domestic pigs. For that, the numbers of enamel incremental markings were recorded in ground sections and compared with crown formation times for the respective teeth derived from literature data on tooth development and eruption in Sus scrofa. Our study revealed that laminations with a daily periodicity are the dominant incremental feature of pig enamel. In wild boar M3s, daily enamel secretion (apposition) rates ranged between a minimum of 6.1 µm in the inner and a maximum of 30.6 µm in the outer enamel.Long-period (supra-daily) incremental markings were present as perikymata at the outer enamel surface (OES). Contrary to the situation in primate enamel, in pig enamel the long-period incremental lines terminating in perikyma grooves were mostly structurally indistinguishable from the daily laminations. Typically, five sub-daily increments were present between successive laminations. The incremental pattern in pig enamel can be misinterpreted if the laminations are mistaken for long-period markings (striae of Retzius) and the sub-daily growth marks for daily prism cross-striations. The findings of the present study demonstrate the critical importance of correctly characterizing the incremental markings and their periodicity in enamel, and caution against an uncritical transfer of the interpretation of the nature of incremental markings in primate enamel to other mammalian taxa.Graphical abstractGraphical abstract for this article
       
  • Ex vivo detection of calcium phosphate and calcium carbonate in rat blood
           serum
    • Abstract: Publication date: Available online 22 November 2018Source: Journal of Structural BiologyAuthor(s): Bao-Di Gou, Yang Liu, Yu-Xi Gao, Kun Tang, Lei Zheng, Yi-Dong Zhao, Tian-Lan Zhang The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42− and HCO3− serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.Graphical abstractThe complexes of calcium phosphate and calcium carbonate in blood serum have physiological activities. Along with the lower-affinity albumin-bound calcium and free calcium, they compose a local chemical system that could play a role in maintaining the calcium level in blood serum.Graphical abstract for this article
       
  • Structural insights into the catalytic mechanism of
           methylthioribose-1-phosphate isomerase
    • Abstract: Publication date: Available online 22 November 2018Source: Journal of Structural BiologyAuthor(s): Prerana Gogoi, Prerana Mordina, Shankar Prasad Kanaujia Methylthioribose-1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of methylthioribose-1-phosphate (MTR-1-P) to methylthioribulose-1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.
       
  • Variation in Human Hair Ultrastructure among Three Biogeographic
           Populations
    • Abstract: Publication date: Available online 22 November 2018Source: Journal of Structural BiologyAuthor(s): Sandra L. Koch, Mark D. Shriver, Nina G. Jablonski Human scalp hairs are often examined microscopically to study the variation and diversity among a range of visible morphological traits. In this study, we focused on the ultrastructure of human scalp hair within its keratinized matrix, emphasizing, the density and distribution of melanosomes, variation in cuticle thickness within populations, and the relationship of hair fiber ultrastructure with biogeographic ancestry. We used transmission electron microscopy (TEM) to visualize hair cross-sections and generate micron-scale resolution images for analysis of particle morphology and the layered hair matrix. Our results revealed considerable variation in all parameters examined, including the relationship of ultrastructure to biogeographic ancestry. Among the three metapopulations studied (European, African, and East Asian), we identified hair cross-sectional shape, cuticle dimensions, and melanosome distribution as traits that reveal statistically significant ancestry-related patterns. This study establishes trait patterns in hair morphology and ultrastructure among three biogeographically defined metapopulations to improve the current understanding of human variation in hair form and establish a foundation for future studies on the genetic and developmental bases of phenotypic variation in hair ultrastructure related to genotype.Graphical abstractGraphical abstract for this article
       
  • Journal of Structural Biology – Paper of the Year 2018
    • Abstract: Publication date: Available online 19 November 2018Source: Journal of Structural BiologyAuthor(s):
       
  • Changing Times: Fluorescence-Lifetime Analysis of Amyloidogenic SF-IAPP
           Fusion Protein
    • Abstract: Publication date: Available online 17 November 2018Source: Journal of Structural BiologyAuthor(s): Olga I Antimonova, Dmitry V Lebedev, Yana A Zabrodskaya, Natalia A Grudinina, Andrey L Timkovsky, Edward Ramsay, Michael M Shavlovsky, Vladimir V Egorov In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils’ detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose.The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM).It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases.We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.
       
  • Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR
           protein from Synechocystis sp. PCC6803
    • Abstract: Publication date: Available online 14 November 2018Source: Journal of Structural BiologyAuthor(s): Patrícia T. Borges, Célia V. Romão, Lígia M. Saraiva, Vera L. Gonçalves, Maria A. Carrondo, Miguel Teixeira, Carlos Frazão Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.Graphical abstractGraphical abstract for this article
       
  • Crystal structure and substrate recognition mechanism of Aspergillus
           oryzae isoprimeverose-producing enzyme
    • Abstract: Publication date: Available online 13 November 2018Source: Journal of Structural BiologyAuthor(s): Tomohiko Matsuzawa, Masahiro Watanabe, Yusuke Nakamichi, Zui Fujimoto, Katsuro Yaoi Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1→6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α)8 TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite −1' played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites −1 and −1', respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.
       
  • Structural and biochemical characterization of citrate binding to AtPPC3,
           a plant-type phosphoenolpyruvate carboxylase from Arabidopsis thaliana
    • Abstract: Publication date: Available online 9 November 2018Source: Journal of Structural BiologyAuthor(s): Matthew B. Connell, Michael J.Y. Lee, Jerry Li, William C. Plaxton, Zongchao Jia Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated cytosolic enzyme situated at a crucial branch point of central plant metabolism. The structure of AtPPC3, a C3 PEPC isozyme of the model plant Arabidopsis thaliana, in complex with the inhibitors aspartate and citrate was solved at 2.2-Å resolution. This represents the first PEPC structure with citrate bound. Aspartate and citrate binding sites are in close proximity (5.1-5.3 Å) and interactions between citrate and specific residues were identified. Citrate functions as a mixed (allosteric) inhibitor as it reduced AtPPC3’s Vmax while increasing Km(PEP) values. The PEP saturation data gave an excellent fit to the mixed inhibition model, yielding Ki and Ki’ (citrate) values of 9.3 and 42.5 mM, respectively. Citrate and aspartate inhibition of AtPPC3 was non-additive, likely due to their closely positioned binding sites, their similar negative charge, and type of binding residues. Fewer interactions and lower affinity for citrate support its observed weaker inhibition of AtPPC3 relative to aspartate. Citrate does not appear to induce further conformational change beyond aspartate owing to the similar structural mechanism of inhibition. AtPPC3 largely exhibits root-specific expression in Arabidopsis, where it is markedly upregulated during stresses such as excessive salinity or nutritional Pi deprivation that necessitate large increases in anaplerotic PEP carboxylation. The cytosolic citrate concentration of potato tubers suggests that AtPPC3’s inhibition by citrate may be physiologically relevant. Our results provide novel insights into the structural basis of allosteric PEPC control and the kinetic effects brought about upon inhibitor binding.Graphical abstractGraphical abstract for this article
       
  • Structural Dissection of Sterol Glycosyltransferase UGT51 from
           Saccharomyces cerevisiae for Substrate Specificity
    • Abstract: Publication date: Available online 2 November 2018Source: Journal of Structural BiologyAuthor(s): Liuqing Chen, Yong Zhang, Yan Feng Sterol glycosyltransferases catalyze the formation of a variety of glycosylated sterol derivatives and are involved in producing a plethora of bioactive natural products. To understand the molecular mechanism of sterol glycosyltransferases, we determined crystal structures of a sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae. The structures of the UGT51 and its complex with uridine diphosphate glucose (UDPG) were solved at resolutions of 2.77 Å and 1.9 Å, respectively. The structural analysis revealed that a long hydrophobic cavity, 9.2 Å in width and 17.6 Å in length located at the N-terminal domain of UGT51, is suitable for the accommodation of sterol acceptor substrates. Furthermore, a short, conserved sequence of S847-M851 was identified at the bottom of the hydrophobic cavity, which might be the steroid binding site and play an important role for the UGT51 catalytic specificity towards sterols. Molecular docking simulations indicated that changed unique interaction network in mutant M7_1 (S801A/L802A/V804A/K812A/E816K/S849A/N892D), with an 1800-fold activity improvement toward an unnatural substrate protopanaxadiol (PPD), might influence its substrate preference. This study reported the first sterol glycosyltransferase structure, providing a molecular blueprint for generating tailored sterol glycosyltransferases as potential catalytic elements in synthetic biology.Graphical abstractGraphical abstract for this article
       
  • Asymmetric protein design from conserved supersecondary structures
    • Abstract: Publication date: Available online 26 October 2018Source: Journal of Structural BiologyAuthor(s): Mohammad ElGamacy, Murray Coles, Andrei Lupas Computational design with supersecondary structures as building blocks has proven effective in the construction of new proteins with controlled geometries. So far, this approach has primarily exploited amplification, effectively harnessing the internal folding propensity of self-compatible fragments to achieve sufficient enthalpy for folding. Here we exploit an interface-driven strategy to depart from the repeat design realm, constructing an asymmetric, globular domain from heterologous supersecondary structures. We report the successful design of a dRP lyase domain fold, which agrees with the experimental NMR structure at atomic accuracy (backbone RMSD of 0.94 Å). Our results show that the residual folding information within conserved fragments, combined with efficient interface-directed sampling, can effectively yield globular proteins with novel sequences and biophysical properties.Graphical abstractGraphical abstract for this article
       
  • Structural and biochemical analysis of a NOT1 MIF4G-like domain of the
           CCR4-NOT complex
    • Abstract: Publication date: Available online 24 October 2018Source: Journal of Structural BiologyAuthor(s): Tobias Raisch, Felix Sandmeir, Oliver Weichenrieder, Eugene Valkov, Elisa Izaurralde The CCR4-NOT complex plays a central role in the regulation of gene expression and degradation of messenger RNAs. The multisubunit complex assembles on the NOT1 protein, which acts as a ‘scaffold’ and is highly conserved in eukaryotes. NOT1 consists of a series of helical domains that serve as docking sites for other CCR4-NOT subunits. We describe a crystal structure of a connector domain of NOT1 from the thermophilic fungus Chaetomium thermophilum (Ct). Comparative structural analysis indicates that this domain adopts a MIF4G-like fold and we have termed it the MIF4G-C domain. Solution scattering studies indicate that the human MIF4G-C domain likely adopts a very similar fold to the Ct MIF4G-C. MIF4G domains have been described to mediate interactions with DEAD-box helicases such as DDX6. However, comparison of the interfaces of the MIF4G-C with the MIF4G domain of NOT1 that interacts with DDX6 reveals key structural differences that explain why the MIF4G-C does not bind DDX6. We further show that the human MIF4G-C does not interact stably with other subunits of the CCR4-NOT complex. The structural conservation of the MIF4G-C domain suggests that it may have an important but presently undefined role in the CCR4-NOT complex.Graphical abstractGraphical abstract for this article
       
  • Crystal structures and biochemical characterization of DNA sliding clamps
           from three Gram-negative bacterial pathogens
    • Abstract: Publication date: Available online 23 October 2018Source: Journal of Structural BiologyAuthor(s): Amy E. McGrath, Alexander P. Martyn, Louise R. Whittell, Fay E. Dawes, Jennifer L. Beck, Nicholas E. Dixon, Michael J. Kelso, Aaron J. Oakley Bacterial sliding clamps bind to DNA and act as protein–protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding clamps via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of β sliding clamps from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli clamps and similar patterns of binding to linear clamp-binding motif peptides. The results suggest that linear motif–sliding clamp interactions are well conserved and an antibiotic targeting the sliding clamp should have broad-spectrum activity against Gram-negative pathogens.Graphical abstractGraphical abstract for this article
       
  • Investigating HCMV entry into host cells by STEM Tomography
    • Abstract: Publication date: Available online 20 October 2018Source: Journal of Structural BiologyAuthor(s): Mohamed E.A. Abdellatif, Christian Sinzger, Paul Walther Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as “Pinocchio particles” were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.Graphical abstractGraphical abstract for this article
       
  • Structure and function of Mycobacterium-specific components of F-ATP
           synthase subunits α and ε
    • Abstract: Publication date: Available online 17 October 2018Source: Journal of Structural BiologyAuthor(s): Nebojša Bogdanović, Lavanya Sundararaman, Neelagandan Kamariah, Anu Tyagi, Shashi Bhushan, Priya Ragunathan, Joon Shin, Thomas Dick, Gerhard Grüber The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system αchi3:β3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:β3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:β3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.Graphical abstractGraphical abstract for this article
       
  • The structure of DcrB, a lipoprotein from Salmonella enterica, reveals
           flexibility in the N-terminal segment of the Mog1p/PsbP-like fold
    • Abstract: Publication date: Available online 16 October 2018Source: Journal of Structural BiologyAuthor(s): Damien M. Rasmussen, Ross W. Soens, Timothy J. Davie, Cody K. Vaneerd, Basudeb Bhattacharyya, John F. May DcrB is an 18 kilodalton lipoprotein that contains a single domain of unknown function. DcrB is found within Enterobacteriaceae, a family of Gram-negative bacteria which includes pathogens that can cause food-borne illness and hospital-acquired infections. In Salmonella enterica serovar Typhimurium, DcrB is up-regulated by conditions that promote the production of known virulence factors. We determined the structure of a truncated form of DcrB from Salmonella to 1.92 Å resolution by X-ray crystallography. This truncated form, DcrBΔ37, contains the entire domain of unknown function but lacks the lipoprotein signal sequence (residues 1–20) as well as residues 21–37. The DcrBΔ37 monomer contains the Mog1p/PsbP-like fold, which is found in functionally diverse proteins in mammals, yeast, plants, and cyanobacteria. Interestingly, DcrBΔ37 crystallized as a domain-swapped homodimer in which the N-terminal β-hairpin extends from one protomer to interact with the core of the second protomer. This domain-swapping indicates that the N-terminal portion of the Mog1p/PsbP-like fold likely has conformational flexibility. Overall, our results provide the first example of an enterobacterial protein that contains the Mog1p/PsbP-like fold and expands knowledge of the structural and phylogenetic diversity of Mog1p/PsbP-like proteins.
       
  • Structural insight into the mechanism of action of antimicrobial peptide
           BMAP-28(1-18) and its analogue mutBMAP18
    • Abstract: Publication date: Available online 15 October 2018Source: Journal of Structural BiologyAuthor(s): Nutan Agadi, Sheeja Vasudevan, Ashutosh Kumar Structural characterization of BMAP-28(1-18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1-18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1-18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1-18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues Ser5, Arg8 and Arg12 of the BMAP-28(1-18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1-18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.Graphical abstractGraphical abstract for this article
       
  • Comparing Cryo-EM Structures
    • Abstract: Publication date: Available online 13 October 2018Source: Journal of Structural BiologyAuthor(s): Catherine L. Lawson, Wah Chiu
       
  • Structural insights into the specificity and catalytic mechanism of
           mycobacterial nucleotide pool sanitizing enzyme MutT2
    • Abstract: Publication date: Available online 9 October 2018Source: Journal of Structural BiologyAuthor(s): Amandeep Singh, Sheikh Mohammad Arif, Pau Biak Sang, Umesh Varshney, M. Vijayan Mis-incorporation of modified nucleotides, such as 5-methyl-dCTP or 8-oxo-dGTP, in DNA can be detrimental to genomic integrity. MutT proteins are sanitization enzymes which function by hydrolyzing such nucleotides and regulating the pool of free nucleotides in the cytoplasm. Mycobacterial genomes have a set of four MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4. Mycobacterial MutT2 hydrolyzes 5m-dCTP and 8-oxo-dGTP to their respective monophosphate products. Additionally, it can hydrolyze canonical nucleotides dCTP and CTP, with a suggested role in sustaining their optimal levels in the nucleotide pool. The structures of M. smegmatis MutT2 and its complexes with cytosine derivates have been determined at resolutions ranging from 1.10 Å to 1.73 Å. The apo enzyme and its complexes with products (dCMP, CMP and 5m-dCMP) crystallize in space group P21212, while those involving substrates (dCTP, CTP and 5m-dCTP) crystallize in space group P21. The molecule takes an α/β/α sandwich fold arrangement, as observed in other MutT homologs. The nucleoside moiety of the ligands is similarly located in all the complexes, while the location of the remaining tail exhibits variability. This is the first report of a MutT2-type protein in complex with ligands. A critical interaction involving Asp116 confers the specificity of the enzyme towards cytosine moieties. A conserved set of enzyme-ligand interactions along with concerted movements of important water molecules provide insights into the mechanism of action.Graphical abstractGraphical abstract for this article
       
  • Crystal structure of a biliverdin-bound phycobiliprotein: interdependence
           of oligomerization and chromophorylation
    • Abstract: Publication date: Available online 1 October 2018Source: Journal of Structural BiologyAuthor(s): Juan Pablo Fuenzalida-Werner, Robert Janowski, Kanuj Mishra, Ina Weidenfeld, Dierk Niessing, Vasilis Ntziachristos, Andre C. Stiel Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.
       
  • Tailored disorder in calcite organization in tergite cuticle of the
           supralittoral isopod Tylos europaeus Arcangeli, 1938
    • Abstract: Publication date: Available online 1 October 2018Source: Journal of Structural BiologyAuthor(s): Bastian H.M. Seidl, Erika Griesshaber, Helge-Otto Fabritius, Christian Reisecker, Sabine Hild, Stefano Taiti, Wolfgang W. Schmahl, Andreas Ziegler The crustacean cuticle forms skeletal elements consisting of chitin-protein fibrils reinforced by amorphous and crystalline calcium carbonate and phosphate minerals. The edges of skeletal elements are of particular interest. They are subject to repeated strain and stress because they form transitions to the arthrodial membranes connecting them. These allow for relative movements of skeletal elements. In this study, we investigate structure, chemical composition, mineral organization and local mechanical properties of the anterior and posterior edges of the tergite cuticle in the conglobating beach isopod Tylos europaeus and compare these with the protective dorsal region of the tergites.The distribution of mineral phases at the edges resembles that of dorsal regions of the tergites. At the transition with the unmineralized arthrodial membrane the calcite containing distal exocuticle is replaced by epicuticular material and the subjacent cuticular layers containing amorphous calcium carbonate become enriched with amorphous calcium phosphate. At the edges, the local elastic modulus and hardness values are significantly lower compared to dorsal regions of the tergite cuticle, for both, the calcite and the amorphous mineral containing layers. The calcite within the tergite cuticle is assembled in different texture patterns: (i) almost random co-orientation, (ii) almost single crystalline calcite, and (iii) a graded organization. Calcite organization and co-orientation strength is highly variable, not only on very few tens of micrometres, but also between regions with different skeletal functionality. Our results show that besides structure and composition, patterns of calcite organization contribute to the hierarchical architecture and functionality of biological composites.Graphical abstractGraphical abstract for this article
       
  • DNP NMR of Biomolecular Assemblies
    • Abstract: Publication date: Available online 29 September 2018Source: Journal of Structural BiologyAuthor(s): Kristaps Jaudzems, Tatyana Polenova, Guido Pintacuda, Hartmut Oschkinat, Anne Lesage Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.
       
  • Map Challenge: Analysis using a Pair Comparison Method based on Fourier
           Shell Correlation
    • Abstract: Publication date: Available online 28 September 2018Source: Journal of Structural BiologyAuthor(s): R. Marabini, M. Kazemi, C.O.S. Sorzano, J.M. Carazo This document presents the analysis performed over the Map Challenge dataset using a new algorithm which we refer to as Pair Comparison Method. The new algorithm, which is described in detail in the text, is able to sort reconstructions based on a figure of merit and assigns a level of significance to the sorting. That is, it shows how likely the sorting is due to chance or if it reflects real differences.
       
  • Structural characterization of the sporulation protein GerM from
           Bacillus subtilis
    • Abstract: Publication date: Available online 25 September 2018Source: Journal of Structural BiologyAuthor(s): Jennyfer Trouve, Ahmed Mohamed, Francisco Leisico, Carlos Contreras-Martel, Bowen Liu, Caroline Mas, David Z. Rudner, Christopher D.A. Rodrigues, Cecile Morlot The Gram-positive bacterium Bacillus subtilis responds to starvation by entering a morphological differentiation process leading to the formation of a highly resistant spore. Early in the sporulation process, the cell asymmetrically divides into a large compartment (the mother cell) and a smaller one (the forespore), which will maturate into a resistant spore. Proper development of the forespore requires the assembly of a multiprotein complex called the SpoIIIA-SpoIIQ complex or “A-Q complex”. This complex involves the forespore protein SpoIIQ and eight mother cell proteins (SpoIIIAA to SpoIIIAH), many of which share structural similarities with components of specialized secretion systems and flagella found in Gram-negative bacteria. The assembly of the A-Q complex across the two membranes that separate the mother cell and forespore was recently shown to require GerM. GerM is a lipoprotein composed of two GerMN domains, a family of domains with unknown function. Here, we report X-ray crystallographic structures of the first GerMN domain of GerM at 1.0 Å resolution, and of the soluble domain of GerM (the tandem of GerMN domains) at 2.1 Å resolution. These structures reveal that GerMN domains can adopt distinct conformations and that the core of these domains display structural similarities with ring-building motifs found in components of specialized secretion system and in SpoIIIA proteins. This work provides an additional piece towards the structural characterization of the A-Q complex.Graphical abstractGraphical abstract for this article
       
  • Direct evidence supporting the existence of a helical dislocation in
           protofilament packing in the intermediate filaments of oxidized trichocyte
           keratin
    • Abstract: Publication date: Available online 21 September 2018Source: Journal of Structural BiologyAuthor(s): R.D. Bruce Fraser, David A.D. Parry The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼74 Å and zb ∼198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called “seam” along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.
       
  • Structural modulation of a periplasmic sugar-binding protein probes into
           its evolutionary ancestry
    • Abstract: Publication date: Available online 20 September 2018Source: Journal of Structural BiologyAuthor(s): Suman Pandey, Prashant S. Phale, Prasenjit Bhaumik Substrate-binding proteins (SBPs) are periplasmic proteins consisting of two α/β domains joined by a hinge region with specificity towards cognate ligands. Based on three-dimensional fold, sugar-specific SBPs have been classified into cluster B and cluster D-I. The analysis of sequences and structures of sugar-binding pocket of cluster D-I SBPs revealed the presence of extra residues on two loops (L1, L2) and a helix (H1) in few members of this family, that binds specifically to monosaccharides. Presence of conserved histidine in L2 and tryptophan in H1 can be considered as the identity marks for the cluster D-I monosaccharide-binding SBPs. A glucose binding protein (ppGBP) from Pseudomonas putida CSV86 was found to contain a structural fold similar to oligosaccharide-binding cluster D-I SBPs, but functionally binds to only glucose due to constriction of its binding pocket mainly by L2 (375-382). ppGBP with partial deletion of L2 (ppGBPΔL2) was created, crystallized and biochemical characterization was performed. Compared to wild type ppGBP, the ppGBPΔL2 structure showed widening of the glucose-binding pocket with ∼80% lower glucose binding. Our results show that the substrate specificity of SBPs can be altered by modulating the size of the binding pocket. Based on this, we propose a sub classification of cluster D-I SBPs into (i) cluster D-I(a)- monosaccharide-binding SBPs and (ii) cluster D-I(b)- oligosaccharide-binding SBPs. This study also provides the direct structural and functional correlation indicating that divergence of proteins may occur through insertions or deletions of sequences in the already existing SBPs leading to evolution at the functional level.Graphical abstractGraphical abstract for this article
       
  • Advances in Instrumentation and Methodology for Solid-State NMR of
           Biological Assemblies
    • Abstract: Publication date: Available online 8 September 2018Source: Journal of Structural BiologyAuthor(s): Rachel W. Martin, John E. Kelly, Jessica I. Kelz Many advances in instrumentation and methodology have furthered the use of solid-state NMR as a technique for determining the structure and studying the dynamics of molecules involved in complex biological assemblies. Solid-state NMR does not require large crystals, has no inherent size limit, and with appropriate isotopic labeling schemes, supports solving one component of a complex assembly at a time. It is complementary to cryo-EM, in that it provides local, atomic-level detail that can be modeled into larger-scale structures. This review focuses on the development of high-field MAS instrumentation and methodology; including probe design, benchmarking strategies, labeling schemes, and experiments that enable the use of quadrupolar nuclei in biomolecular NMR. Current challenges facing solid-state NMR of biological assemblies and new directions in this dynamic research area are also discussed.
       
  • Polyserine repeats promote coiled coil-mediated fibril formation and
           length-dependent protein aggregation
    • Abstract: Publication date: Available online 6 September 2018Source: Journal of Structural BiologyAuthor(s): Elena Lilliu, Veronica Villeri, Ilaria Pelassa, Federico Cesano, Domenica Scarano, Ferdinando Fiumara Short polyserine (polyS) repeats are frequently found in proteins and longer ones are produced in neurological disorders such as Huntington disease (HD) owing to translational frameshifting or non-ATG-dependent translation, alongside with polyglutamine (polyQ) and polyalanine (polyA) repeats, forming intracellular aggregates. However, the physiological and pathological structures of polyS repeats are not clearly understood. Early studies highlighted their structural versatility, similar to other homopolymers whose conformation is influenced by the surrounding protein context. As polyS stretches are frequently near polyQ and polyA repeats, which can be part of coiled coil (CC) structures, and the frameshift-derived polyS repeats in HD directly flank CC heptads important for aggregation, we investigate here the structural and aggregation properties of polyS in the context of CC structures. We have taken advantage of peptide models, previously used to study polyQ and polyA in CCs, in which we inserted polyS repeats of variable length and studied them in comparison with polyQ and polyA peptides. We found that polyS peptides promote CC-mediated polymerization and fibrillization as revealed by circular dichroism, chemical crosslinking, and atomic force microscopy. Furthermore, they promote CC-based, length-dependent, intracellular aggregation, which is negligible with 7 and widespread with 49 serines. These findings show that polyS repeats can participate in the formation of CCs, as previously found for polyQ and polyA, conferring to peptides peculiar structural properties, with aggregation kinetics that are intermediate between those of polyA and polyQ CCs, and contribute to an overall structural definition of the pathophysiogical roles of homopolymeric repeats in CC structures.
       
  • New software tools in EMAN2 inspired by EMDatabank map challenge
    • Abstract: Publication date: Available online 4 September 2018Source: Journal of Structural BiologyAuthor(s): James M. Bell, Muyuan Chen, Tunay Durmaz, Adam C. Fluty, Steven J. Ludtke EMAN2 is an extensible software suite with complete workflows for performing high-resolution single particle analysis, 2-D and 3-D heterogeneity analysis, and subtomogram averaging, among other tasks. Participation in the recent CryoEM Map Challenge sponsored by the EMDatabank led to a number of significant improvements to the single particle analysis process in EMAN2. A new convolutional neural network particle picker was developed, which dramatically improves particle picking accuracy for difficult data sets. A new particle quality metric capable of accurately identifying “bad” particles with a high degree of accuracy, no human input, and a negligible amount of additional computation, has been introduced, and this now serves as a replacement for earlier human-biased methods. The way 3-D single particle reconstructions are filtered has been altered to be more comparable to the filter applied in several other popular software packages, dramatically improving the appearance of sidechains in high-resolution structures. Finally, an option has been added to perform local resolution-based iterative filtration, resulting in local resolution improvements in many maps.
       
  • Structural Flexibility in the Helicobacter pylori Peptidyl-prolyl cis,
           trans-Isomerase HP0175 is achieved through an Extension of the Chaperone
           Helices
    • Abstract: Publication date: Available online 1 September 2018Source: Journal of Structural BiologyAuthor(s): Ayat Yaseen, Gerald Audette Helicobacter pylori infects the gastric epithelium of half the global population, where infections can persist into adenocarcinomas and peptic ulcers. H. pylori secretes several proteins that lend to its pathogenesis and survival including VacA, CagA, γ-glutamyltransferase and HP0175. HP0175, also known as HpCBF2, classified as a peptidyl-prolyl cis,trans-isomerase, has been shown to induce apoptosis through a cascade of mechanisms initiated though its interaction with toll like receptor 4 (TLR4). Here, we report the structure of apo-HP0175 at 2.09 Å with a single monomer in the asymmetric unit. Chromatographic, light scattering and mass spectrometric analysis of HP0175 in solution indicate that the protein is mainly monomeric under low salt conditions, while increasing ionic interactions facilitates protein dimerization. A comparison of the apo-HP0175 structure to that of the indole-2-carboxylic acid-bound form shows movement of the N- and C-terminal helices upon interaction of the catalytic residues in the binding pocket. Helix extension of the N/C chaperone domains between apo and I2CA-bound HP0175 supports previous findings in parvulin PPIases for their role in protein stabilization (and accommodation of variable protein lengths) of those undergoing catalysis.
       
  • Flexibility of the sec13/31 cage is influenced by the sec31 C-terminal
           disordered domain
    • Abstract: Publication date: Available online 30 August 2018Source: Journal of Structural BiologyAuthor(s): Mohammadreza Paraan, Nilakshee Bhattacharya, Vladimir N. Uversky, Scott M. Stagg In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.
       
  • A new algorithm for high-resolution reconstruction of single particles by
           electron microscopy
    • Abstract: Publication date: Available online 24 August 2018Source: Journal of Structural BiologyAuthor(s): C.O.S. Sorzano, J. Vargas, J.M. de la Rosa-Trevín, A. Jiménez, D. Maluenda, R. Melero, M. Martínez, E. Ramírez-Aportela, P. Conesa, J.L. Vilas, R. Marabini, J.M. Carazo The Map Challenge organized by the Electron Microscopy Data Bank has prompted the development of an Xmipp high resolution reconstruction protocol (which we will refer to as highres) that is integrated in the software platform Scipion. In this work we describe the details of the image angular alignment and map reconstruction steps in our new method. This algorithm is similar to the standard projection matching approach with some important modifications, especially in the area of detecting significant features in the reconstructed volume. We show that the new method is able to produce higher resolution maps than the current de facto standard as measured by the Fourier Shell Correlation, the Monogenic Local Resolution and EMRinger.
       
  • APPLE Picker: Automatic Particle Picking, a Low-Effort Cryo-EM Framework
    • Abstract: Publication date: Available online 19 August 2018Source: Journal of Structural BiologyAuthor(s): Ayelet Heimowitz, Joakim Andén, Amit Singer Particle picking is a crucial first step in the computational pipeline of single-particle cryo-electron microscopy (cryo-EM). Selecting particles from the micrographs is difficult especially for small particles with low contrast. As high-resolution reconstruction typically requires hundreds of thousands of particles, manually picking that many particles is often too time-consuming. While template-based particle picking is currently a popular approach, it may suffer from introducing manual bias into the selection process. In addition, this approach is still somewhat time-consuming. This paper presents the APPLE (Automatic Particle Picking with Low user Effort) picker, a simple and novel approach for fast, accurate, and template-free particle picking. This approach is evaluated on publicly available datasets containing micrographs of β-Galactosidase, T20S proteasome, 70S ribosome and keyhole limpet hemocyanin projections.
       
  • Structural behavior of keratin-associated protein 8.1 in human hair as
           revealed by a monoclonal antibody
    • Abstract: Publication date: Available online 18 August 2018Source: Journal of Structural BiologyAuthor(s): Hiroki Akiba, Emina Ikeuchi, Ganbat Javkhlan, Hiroki Fujikawa, Osamu Arai-Kusano, Hiroko Iwanari, Makoto Nakakido, Takao Hamakubo, Yutaka Shimomura, Kouhei Tsumoto Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein’s roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1in native conformation, suggesting that structural features of KAP8.1 vary among species.Graphical abstractA) Immunohistological image of E4304-stained hair tissue from healthy Japanese donors. Red: E4304-stained KAP8.1, blue: DAPI-stained cell nuclei. B) Images of in situ hybridization of KRTAP8-1-mRNA in human hair samples. KZ, keratinizing zone; HB, hair bulb. C) Model structure of KAP8.1.Graphical abstract for this article
       
  • The analysis of subtle internal communications through mutation studies in
           periplasmic metal uptake protein CLas-ZnuA2
    • Abstract: Publication date: Available online 17 August 2018Source: Journal of Structural BiologyAuthor(s): Gunjan Saini, Nidhi Sharma, Vikram Dalal, Ashish Warghane, Dilip Kumar Ghosh, Pravindra Kumar, Ashwani Kumar Sharma The subtle internal communications through an intricate network of interactions play a key role in metal-binding and release in periplasmic metal uptake proteins of cluster A-I family, a component of ABC transport system. These proteins have evolved different mechanisms of metal-binding and release through sequence and thereby structure-function divergence. The CLas-ZnuA2 from Candidatus Liberibacter asiaticus (CLA), in previous studies, showed a lower metal-binding affinity. The subtle communications within and between domains from crystal structure analysis revealed that protein seems to prefer a metal-free state. The unique features of CLas-ZnuA2 included a highly restrained loop L3 and presence of a proline in linker helix. In present work, S38A and Y68F mutants were studied as they play an important role during metal-binding in CLas-ZnuA2. The mutations in linker helix could not be studied as the expressed protein was not soluble and in most cases degraded with time. The crystal structure analysis of (S38A and Y68F) mutants in metal-free and metal-bound forms showed variations in interactions, an increase in number of alternate conformations and distortions in secondary structure elements, despite a similar overall structure, suggesting alterations in internal communications. The results suggested that any change in critical residues could alter the subtle internal communications and result in disturbing the fine-tuned structure required for optimal functioning.Graphical abstractThe superposition involving metal-free states of S38A CLas-ZnuA2 (green) and wild-type CLas-ZnuA2 (pink) (PDBID:4UDN) showing variation in secondary structure elements in C- domain is shown.Graphical abstract for this article
       
  • 752Structural and functional analyses of calcium ion response factors in
           the mantle of Pinctada fucata
    • Abstract: Publication date: Available online 17 August 2018Source: Journal of Structural BiologyAuthor(s): Akihiro Matsuura, Ko Yoshimura, Hiroyuki Kintsu, Takashi Atsumi, Yasushi Tsuchihashi, Takeshi Takeuchi, Noriyuki Satoh, Lumi Negishi, Shohei Sakuda, Tomiko Asakura, Yuki Imura, Etsuro Yoshimura, Michio Suzuki The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.Graphical abstractGraphical abstract for this article
       
  • Conformational flexibility of pore loop-1 gives insights into substrate
           translocation by the AAA+ protease FtsH
    • Abstract: Publication date: Available online 14 August 2018Source: Journal of Structural BiologyAuthor(s): Matthias Uthoff, Ulrich Baumann Two crystal structures of a transmembrane helix-lacking FtsH construct from Aquifex aeolicus have been determined at 2.9 Å and 3.3 Å resolution in space groups R32 and P312, respectively. Both structures are virtually identical despite different crystal packing contacts. In both structures, the FtsH hexamer is created from two different subunits of the asymmetric unit by the three-fold symmetry of the crystals. Similar to other published structures, all subunits are loaded with ADP and the two subunit in the asymmetric unit resemble the already known open and closed conformations. Within the ATPase cycle while the whole subunit switches from the opened to the closed state, pore loop-1 interacts with the substrate and translocates it into the proteolytic chamber. Unique to our models is a presumably inactive conformation of the pore loop which allows the closed conformation to switch back to the opened state without pushing the substrate out again. Our structures give further insights on how this new pore loop conformation is induced and how it is linked to the intersubunit signalling network.Graphical abstractGraphical abstract for this article
       
  • The First Single Particle Analysis Map Challenge: A Summary of the
           Assessments
    • Abstract: Publication date: Available online 13 August 2018Source: Journal of Structural BiologyAuthor(s): J. Bernard Heymann, Roberto Marabini, Mohsen Kazemi, Carlos Oscar S. Sorzano, Maya Holmdahl, Joshua H. Mendez, Scott M. Stagg, Slavica Jonic, Eugene Palovcak, Jean-Paul Armache, Jianhua Zhao, Yifan Cheng, Grigore Pintilie, Wah Chiu, Ardan Patwardhan, Jose-Maria Carazo The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.
       
  • Building Atomic Models Based on Near Atomic Resolution cryoEM Maps with
           Existing Tools
    • Abstract: Publication date: Available online 13 August 2018Source: Journal of Structural BiologyAuthor(s): Iris Yu, Lisa Nguyen, Jaycob Avaylon, Kevin Wang, Mason Lai, Z. Hong Zhou The EMDataBank Validation Challenge was a challenging task for students newly introduced to the cryoEM and molecular modeling fields. However, the competition provided an effective space for student modelers to discover and explore the potentials of atomic modeling and refinement by practicing on published atomic structures. Here, by employing manual molecular modeling programs such as Coot, Phenix, and Chimera, we have regularized and improved three targets. The T20S proteasome and TRPV1 ion channel allowed us to broaden our understanding of our techniques while the 70S ribosome served as a challenge to test the limits of our abilities. We were successful in our efforts to improve each of our models and provide here our cohesive methodology for de novo modeling with and without homology models, which may serve as a starting point for other undergraduates and researchers just entering the realm of cryoEM. Additionally, we provide some constructive criticism to facilitate the introduction of said undergraduates and researchers into cryoEM in the future.
       
  • The 3-D structure of VNG0258H/RosR – a haloarchaeal DNA-binding
           protein in its ionic shell
    • Abstract: Publication date: Available online 12 August 2018Source: Journal of Structural BiologyAuthor(s): Nitzan Kutnowski, Hagay Shmuely, Idit Dahan, Fania Shmulevich, Geula Davidov, Anat Shahar, Jerry Eichler, Raz Zarivach, Boaz Shaanan Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in the halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus have significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences with implications to protein-DNA interactions in hyper-saline environments.Graphical abstractGraphical abstract for this article
       
  • Assessment of detailed conformations suggests strategies for improving
           cryoEM models: Helix at lower resolution, ensembles, pre-refinement
           fixups, and validation at multi-residue length scale
    • Abstract: Publication date: Available online 11 August 2018Source: Journal of Structural BiologyAuthor(s): Jane S. Richardson, Christopher J. Williams, Lizbeth L. Videau, Vincent B. Chen, David C. Richardson We find that the overall quite good methods used in the CryoEM Model Challenge could still benefit greatly from several strategies for improving local conformations. Our assessments primarily use validation criteria from the MolProbity web service. Those criteria include MolProbity's all-atom contact analysis, updated versions of standard conformational validations for protein and RNA, plus two recent additions: first, flags for cis-nonPro and twisted peptides, and second, the CaBLAM system for diagnosing secondary structure, validating Cα backbone, and validating adjacent peptide CO orientations in the context of the Cα trace. In general, automated ab initio building of starting models is quite good at backbone connectivity but often fails at local conformation or sequence register, especially at poorer than 3.5 Å resolution. However, we show that even if criteria (such as Ramachandran or rotamer) are explicitly restrained to improve refinement behavior and overall validation scores, automated optimization of a deposited structure seldom corrects specific misfittings that start in the wrong local minimum, but just hides them. Therefore, local problems should be identified, and as many as possible corrected, before starting refinement. Secondary structures are confusing at 3–4 Å but can be better recognized at 6–8 Å. In future model challenges, specific steps being tested (such as segmentation) and the required documentation (such as PDB code of starting model) should each be explicitly defined, so competing methods on a given task can be meaningfully compared. Individual local examples are presented here, to understand what local mistakes and corrections look like in 3D, how they probably arise, and what possible improvements to methodology might help avoid them. At these resolutions, both structural biologists and end-users need meaningful estimates of local uncertainty, perhaps through explicit ensembles. Fitting problems can best be diagnosed by validation that spans multiple residues; CaBLAM is such a multi-residue tool, and its effectiveness is demonstrated.
       
  • 3D micro structural analysis of human cortical bone in paired femoral
           diaphysis, femoral neck and radial diaphysis
    • Abstract: Publication date: Available online 11 August 2018Source: Journal of Structural BiologyAuthor(s): Rémy Gauthier, Max Langer, Hélène Follet, Cécile Olivier, Pierre-Jean Gouttenoire, Lukas Helfen, Frédéric Rongiéras, David Mitton, Françoise Peyrin Human bone is known to adapt to its mechanical environment in a living body. Both its architecture and microstructure may differ between weight-bearing and non-weight-bearing bones. The aim of the current study was to analyze in three dimensions, the morphology of the multi-scale porosities on human cortical bone at different locations. Eight paired femoral diaphyses, femoral necks, and radial diaphyses were imaged using Synchrotron Radiation µCT with a 0.7 µm isotropic voxel size. The spatial resolution facilitates the investigation of the multiscale porosities of cortical bone, from the osteonal canals system down to the osteocyte lacunar system. Our results showed significant differences in the microstructural properties, regarding both osteonal canals and osteocytes lacunae, between the different anatomical locations. The radius presents significantly lower osteonal canal volume fraction and smaller osteonal canals than the femoral diaphysis or neck. Osteocytes lacunae observed in the radius are significantly different in shape than in the femur, and lacunar density is higher in the femoral neck. These results show that the radius, a non-weight-bearing bone, is significantly different in terms of its microstructure from a weight-bearing bone such as the femur. This implies that the cortical bone properties evaluated on the femoral diaphysis, the main location studied within the literature, cannot be generalized to other anatomical locations.
       
  • Rapid near-atomic resolution single-particle 3D reconstruction with SIMPLE
    • Abstract: Publication date: Available online 6 August 2018Source: Journal of Structural BiologyAuthor(s): Cyril F. Reboul, Simon Kiesewetter, Michael Eager, Matthew Belousoff, Tiangang Cui, Hans De Sterck, Dominika Elmlund, Hans Elmlund Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.
       
  • Conformational plasticity of the response regulator CpxR, a key player in
           Gammaproteobacteria virulence and drug-resistance
    • Abstract: Publication date: Available online 4 August 2018Source: Journal of Structural BiologyAuthor(s): Ariel E. Mechaly, Ahmed Haouz, Nathalie Sassoon, Alejandro Buschiazzo, Jean-Michel Betton, Pedro M. Alzari The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.
       
  • Contributions of different modules of the plasminogen-binding
           
    • Abstract: Publication date: Available online 30 July 2018Source: Journal of Structural BiologyAuthor(s): Cunjia Qiu, Yue Yuan, Jaroslav Zajicek, Zhong Liang, Rashna D. Balsara, Teresa Brito-Robionson, Shaun W. Lee, Victoria A. Ploplis, Francis J. Castellino Group A Streptococcus pyogenes (GAS) is a causative agent of pharyngeal and dermal infections in humans. A major virulence determinant of GAS is its dimeric signature fibrillar M-protein (M-Prt), which is evolutionarily designed in modules, ranging from a hypervariable extracellular N-terminal region to a progressively more highly conserved C-terminus that is covalently anchored to the cell wall. Of the>250 GAS isolates classified, only the subset of skin-trophic Pattern D strains expresses a specific serotype of M-Prt, PAM, that directly binds to host human plasminogen (hPg) via its extracellular NH2-terminal variable A-domain region. This interaction allows these GAS strains to accumulate components of the host fibrinolytic system on their surfaces to serve extracellular functions. While structure-function studies have been accomplished on M-Prts from Pattern A-C GAS isolates with different direct ligand binding properties from PAM, much less is known regarding the structure-function relationships of PAM-type M-Prts, particularly their dimerization determinants. To examine these questions, PAMs from seven GAS strains with sequence variations in the NH2-terminal ligand binding domains, as well as truncated versions of PAM, were designed and studied. The results from bioinformatic and biophysical analyses show that the different domains of PAM are disparately engaged in dimerization. From these data, we propose an experimentally-based model for PAM secondary and quaternary structures that is highly dependent on the conserved helical C-terminal C-D-domains. In addition, while the N-terminal regions of PAMs are variable in sequence, the binding properties of hPg and its activated product, plasmin, to the A-domain, remain intact.Graphical abstractGraphical abstract for this article
       
  • High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image
           Shift
    • Abstract: Publication date: Available online 25 July 2018Source: Journal of Structural BiologyAuthor(s): Anchi Cheng, Edward T. Eng, Lambertus Alink, William J. Rice, Kelsey D. Jordan, Laura Y. Kim, Clinton S. Potter, Bridget Carragher Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images.For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (+/- 0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error; with π/4 phase error considered as the worst that can be accepted. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit.To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.
       
  • Studying intact bacterial peptidoglycan by proton-detected NMR
           spectroscopy at 100 kHz MAS frequency
    • Abstract: Publication date: Available online 19 July 2018Source: Journal of Structural BiologyAuthor(s): Catherine Bougault, Isabel Ayala, Waldemar Vollmer, Jean-Pierre Simorre, Paul Schanda The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeniety, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling.
       
  • Map Challenge Assessment: Fair comparison of single particle cryoEM
           reconstructions
    • Abstract: Publication date: Available online 17 July 2018Source: Journal of Structural BiologyAuthor(s): J. Bernard Heymann Cryo-electron microscopy (cryoEM) is capable of achieving near-atomic resolution of biomolecular structures due to recent advances in hardware. Despite the long history of image processing software development for cryoEM, uncertainty about best practices and validation remains. The Map Challenge was therefore designed to test the current state of single particle reconstruction. As the first such challenge, the participants were given the freedom to analyze the cases in whichever way they wanted. Therefore, the maps submitted feature different sizes, sampling and orientations, making assessment non-trivial. To be fair, I developed a method to pose all maps in each case in the same configuration with minimal interpolation. I assess the quality of these maps by visual inspection and Fourier shell correlation (FSC). Comparing the even-odd FSC with an FSC calculated against a reference structure analysis, I conclude that the quality of the maps related more to the user than to other factors, such as the software package used. Poor quality maps suffer either from lack of data or poor choices made by the user. Some maps appear significantly better than a reference or consensus of other maps, indicating overfitting. Best practices to avoid problems include an understanding of the effects of reference map modifications on particle image alignment, and generating appropriate masks. Ultimately, none of the issues revealed in the Map Challenge is insurmountable, as underscored by the excellent quality of reconstructions achieved by a significant number of participants.
       
  • Crystal structure of saposin D in an open conformation
    • Abstract: Publication date: Available online 17 July 2018Source: Journal of Structural BiologyAuthor(s): Ahmad Gebai, Alexei Gorelik, Bhushan Nagar Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity. The structure of saposin D was previously determined in an inactive conformation, revealing a monomeric, closed and compact form. Here, we present the crystal structure of the open, V-shaped form of saposin D. The overall shape is similar to the open conformation found in other saposins with slight differences in the angles between the α-helices. The structure forms a dimer that serves to stabilize the hydrophobic surface exposed in the open form, which results in an internal, hydrophobic cavity that could be used to carry extracted membrane lipids.
       
  • Modulation of calcium oxalate dihydrate growth by phosphorylated
           osteopontin peptides
    • Abstract: Publication date: Available online 17 July 2018Source: Journal of Structural BiologyAuthor(s): Yung-Ching Chien, Ahmad Mansouri, Wenge Jiang, Saeed R. Khan, Jeffrey J. Gray, Marc D. McKee Osteopontin (OPN) is a significant component of kidney stone matrix and a key modulator of stone formation. Here, we investigated the effects of different phosphorylated states of a synthesized peptide of OPN (the ASARM peptide; acidic, serine- and aspartate-rich motif) on calcium oxalate dihydrate (COD) crystals, a major mineral phase of kidney stones. In vitro, phosphorylated OPN-ASARM peptides strongly inhibited COD crystal growth in solution as compared to the nonphosphorylated state, with increasing inhibitory potency correlating with the degree of peptide phosphorylation. Scanning electron microscopy revealed that the inhibition from the phosphopeptides resulted in distinctive, rosette-like crystal aggregates called spherulites. The OPN-ASARM peptides preferentially bound and specifically inhibited the {1 1 0} crystallographic faces of COD, as identified by combining atomic force microscopy and computational simulation approaches. These {1 1 0} surfaces of COD have high lattice calcium occupancy (exposure), providing preferential binding sites for the highly acidic peptides; binding and inhibition by OPN-ASARM peptides at the {1 1 0} faces led to crystal aggregation and intergrowth. The crystal spherulite formations obtained in vitro when using the most phosphorylated form of OPN-ASARM peptide at a high concentration, resembled crystal morphologies observed in vivo in a rat model of urolithiasis, in which crystal deposits in the kidney contain abundant OPN as revealed by immunogold labeling. A mechanistic model for spherulite formation is proposed based on the symmetry and crystallographic structure of COD, where the phosphate groups of OPN-ASARM bind to calcium atoms at [1 1 1] step risers on the COD {1 1 0} surface, inducing the periodic emergence of new COD crystals to form spherulites.
       
  • Solid-state NMR analysis of the sodium pump Krokinobacter
           rhodopsin 2 and its H30A mutant
    • Abstract: Publication date: Available online 4 June 2018Source: Journal of Structural BiologyAuthor(s): Jagdeep Kaur, Clara Nassrin Kriebel, Peter Eberhardt, Orawan Jakdetchai, Alexander J. Leeder, Ingrid Weber, Lynda J. Brown, Richard C.D. Brown, Johanna Becker-Baldus, Christian Bamann, Josef Wachtveitl, Clemens Glaubitz Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.
       
  • Spectral comparisons of mammalian cells and intact organelles by
           solid-state NMR
    • Abstract: Publication date: Available online 30 May 2018Source: Journal of Structural BiologyAuthor(s): Sabrina H. Werby, Lynette Cegelski Whole-cell protein profiling, spatial localization, and quantification of activities such as gene transcription and protein translation are possible with modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within a cell – capturing the relative amounts of protein and ribosomal RNA levels and lipid content simultaneously – would require extractions and purifications with caveats due to isolation yields and detection methods. A holistic view of cellular composition would aid in the study of cellular composition and function. Here, solid state NMR is used to identify 13C NMR signatures for cellular organelles in HeLa cells without the use of any isotopic labeling. Comparisons are made with carbon spectra of subcellular assemblies including DNA, lipids, ribosomes, nuclei and mitochondria. Whole-cell comparisons are made with different mammalian cells lines, with red blood cells that lack nuclei and organelles, and with Gram-negative and Gram-positive bacteria. Furthermore, treatment of mammalian cells with cycloheximide, a commonly used protein synthesis inhibitor, revealed unanticipated changes consistent with a significant increase in protein glycosylation, obvious at the whole cell level. Thus, we demonstrate that solid-state NMR serves as a unique analytical tool to catalog and compare the ratios of distinct carbon types in cells and serves as a discovery tool to reveal the workings of inhibitors such as cycloheximide on whole-cell biochemistry.
       
  • Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils
           Probed by Paramagnetic Solid-State NMR Spectroscopy
    • Abstract: Publication date: Available online 18 April 2018Source: Journal of Structural BiologyAuthor(s): Darryl Aucoin, Yongjie Xia, Theint Theint, Philippe S. Nadaud, Krystyna Surewicz, Witold K. Surewicz, Christopher P. Jaroniec The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.
       
  • Comparison of the 3D Structures of Mouse and Human α-Synuclein Fibrils by
           Solid-State NMR and STEM
    • Abstract: Publication date: Available online 17 April 2018Source: Journal of Structural BiologyAuthor(s): Songhwan Hwang, Pascal Fricke, Maximilian Zinke, Karin Giller, Joseph S. Wall, Dietmar Riedel, Stefan Becker, Adam Lange Intra-neuronal aggregation of α-synuclein into fibrils is the molecular basis for α -synucleinopathies, such as Parkinson’s disease. The atomic structure of human α -synuclein (hAS) fibrils was recently determined by Tuttle et al. using solid-state NMR (ssNMR). The previous study found that hAS fibrils are composed of a single protofilament. Here, we have investigated the structure of mouse α -synuclein (mAS) fibrils by STEM and isotope-dilution ssNMR experiments. We found that in contrast to hAS, mAS fibrils consist of two or even three protofilaments which are connected by rather weak interactions in between them. Although the number of protofilaments appears to be different between hAS and mAS, we found that they have a remarkably similar secondary structure and protofilament 3D structure as judged by secondary chemical shifts and intra-molecular distance restraints. We conclude that the two mutant sites between hAS and mAS (positions 53 and 87) in the fibril core region are crucial for determining the quaternary structure of α -synuclein fibrils.
       
  • Amyloid structure of high-order assembly of Leucine-rich amelogenin
           revealed by solid-state NMR
    • Abstract: Publication date: Available online 28 March 2018Source: Journal of Structural BiologyAuthor(s): Cheng-Wei Ma, Jing Zhang, Xing-Qi Dong, Jun-Xia Lu High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with β-sheet secondary structure (residues 12–27) and the β-sheet segment would further assemble into amyloid-like structures.
       
  • Interplay between membrane curvature and protein conformational
           equilibrium investigated by solid-state NMR
    • Abstract: Publication date: Available online 1 March 2018Source: Journal of Structural BiologyAuthor(s): Shu Y. Liao, Myungwoon Lee, Mei Hong Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.
       
  • Coiled-coil oligomerization controls localization of the plasma membrane
           REMORINs
    • Abstract: Publication date: Available online 23 February 2018Source: Journal of Structural BiologyAuthor(s): Denis Martinez, Anthony Legrand, Julien Gronnier, Marion Decossas, Paul Gouguet, Olivier Lambert, Mélanie Berbon, Loris Verron, Axelle Grélard, Veronique Germain, Antoine Loquet, Sébastien Mongrand, Birgit Habenstein REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.
       
 
 
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