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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  [3162 journals]
  • 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
    • 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
    • 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
    • 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
  • Reconstituting the formation of hierarchically porous silica patterns
           using diatom biomolecules
    • Abstract: Publication date: Available online 29 July 2018Source: Journal of Structural BiologyAuthor(s): Damian Pawolski, Christoph Heintze, Ingo Mey, Claudia Steinem, Nils Kröger The genetically-controlled formation of complex-shaped inorganic materials by living organisms is an intriguing phenomenon. It illustrates our incomplete understanding of biological morphogenesis and demonstrates the feasibility of ecologically benign routes for materials technology. Amorphous SiO2 (silica) is taxonomically the most widespread biomineral, with diatoms, a large group of single-celled microalgae, being the most prolific producers. Silica is the main component of diatom cell walls, which exhibit species-specific patterns of pores that are hierarchically arranged and endow the material with advantageous properties. Despite recent advances in characterizing diatom biomolecules involved in biosilica morphogenesis, the mechanism of this process has remained controversial. Here we describe the in vitro synthesis of diatom-like, porous silica patterns using organic components that were isolated from biosilica of the diatom Cyclotella cryptica. The synthesis relies on the synergism of soluble biomolecules (long-chain polyamines and proteins) with an insoluble nanopatterned organic matrix. Biochemical dissection of the process revealed that the long-chain polyamines rather than the proteins are essential for efficient in vitro synthesis of the hierarchically porous silica patterns. Our results support the organic matrix hypothesis for morphogenesis of diatom biosilica and introduce organic matrices from diatoms as a new tool for the synthesis of meso- to microporous inorganic materials.
  • High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image
    • 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.
  • Improved sample dispersion in cryo-EM using
           “perpetually-hydrated” graphene oxide flakes
    • Abstract: Publication date: Available online 18 July 2018Source: Journal of Structural BiologyAuthor(s): Martin Cheung, Hidehito Adaniya, Cathal Cassidy, Masao Yamashita, Kun-Lung Li, Seita Taba, Tsumoru Shintake For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using “perpetually-hydrated” graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.
  • Map Challenge Assessment: Fair comparison of single particle cryoEM
    • 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.
  • Evaluation System and Web Infrastructure for the Second Cryo-EM Model
    • Abstract: Publication date: Available online 12 July 2018Source: Journal of Structural BiologyAuthor(s): Andriy Kryshtafovych, Paul D. Adams, Catherine L. Lawson, Wah Chiu An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at
  • A simple and robust procedure for preparing graphene-oxide cryo-EM grids
    • Abstract: Publication date: Available online 11 July 2018Source: Journal of Structural BiologyAuthor(s): Eugene Palovcak, Feng Wang, Shawn Q. Zheng, Zanlin Yu, Sam Li, Miguel Betegon, David Bulkley, David A. Agard, Yifan Cheng Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface. Additionally, the signal of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.
  • Optimal data-driven parameterization of coiled coils
    • Abstract: Publication date: Available online 9 July 2018Source: Journal of Structural BiologyAuthor(s): Dmytro Guzenko, Sergei V. Strelkov α-helical coiled coils (CCs) represent an important, highly regular protein folding motif. To date, many thousands of CC structures have been determined experimentally. Their geometry is usually modelled by theoretical equations introduced by F. Crick that involve a predefined set of parameters. Here we have addressed the problem of efficient CC parameterization from scratch by performing a statistical evaluation of all available CC structures. The procedure is based on the principal component analysis and yields a minimal set of independent parameters that provide for the reconstruction of the complete CC structure at a required precision. The approach is successfully validated on a set of canonical parallel CC dimers. Its applications include all cases where an efficient sampling of the CC geometry is important, such as for solving the phase problem in crystallography.
  • Single particle reconstruction and validation using Bsoft for the map
    • Abstract: Publication date: Available online 5 July 2018Source: Journal of Structural BiologyAuthor(s): J. Bernard Heymann The Bsoft package is aimed at processing electron micrographs for the determination of the three-dimensional structures of biological specimens. Recent advances in hardware allow us to solve structures to near atomic resolution using single particle analysis (SPA). The Map Challenge offered me an opportunity to test the ability of Bsoft to produce reconstructions from cryo-electron micrographs at the best resolution. I also wanted to understand what needed to be done to work towards full automation with validation. Here, I present two cases for the Map Challenge using Bsoft: ß-galactosidase and GroEL. I processed two independent subsets in each case with resolution-limited alignment. In both cases the reconstructions approached the expected resolution within a few iterations of alignment. I further validated the results by coherency-testing: i.e., that the reconstructions from real particles give better resolutions than reconstructions from the same number of aligned noise images. The key operations requiring attention for full automation are: particle picking, faster accurate alignment, proper mask generation, appropriate map sharpening, and understanding the amount of data needed to reach a desired resolution.
  • Routine Determination of Ice Thickness for Cryo-EM Grids
    • Abstract: Publication date: Available online 4 July 2018Source: Journal of Structural BiologyAuthor(s): William J. Rice, Anchi Cheng, Alex J. Noble, Edward T. Eng, Laura Y. Kim, Bridget Carragher, Clinton S. Potter Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.
  • Elemental composition and ultrafine structure of the skeleton in
           shell-bearing protists—A case study of phaeodarians and radiolarians
    • Abstract: Publication date: Available online 3 July 2018Source: Journal of Structural BiologyAuthor(s): Yasuhide Nakamura, Izumi Iwata, Rie S. Hori, Naomi Uchiyama, Akihiro Tuji, Masaki J. Fujita, Daiske Honda, Hiroaki Ohfuji Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.
  • Variability of the core geometry in parallel coiled-coil bundles
    • Abstract: Publication date: Available online 2 July 2018Source: Journal of Structural BiologyAuthor(s): Krzysztof Szczepaniak, Jan Ludwiczak, Aleksander Winski, Stanislaw Dunin-Horkawicz In protein modelling and design, an understanding of the relationship between sequence and structure is essential. Using parallel, homotetrameric coiled-coil structures as a model system, we demonstrated that machine learning techniques can be used to predict structural parameters directly from the sequence. Coiled coils are regular protein structures, which are of great interest as building blocks for assembling larger nanostructures. They are composed of two or more alpha-helices wrapped around each other to form a supercoiled bundle. The coiled-coil bundles are defined by four basic structural parameters: topology (parallel or antiparallel), radius, degree of supercoiling, and the rotation of helices around their axes. In parallel coiled coils the latter parameter, describing the hydrophobic core packing geometry, was assumed to show little variation. However, we found that subtle differences between structures of this type were not artifacts of structure determination and could be predicted directly from the sequence. Using this information in modelling narrows the structural parameter space that must be searched and thus significantly reduces the required computational time. Moreover, the sequence-structure rules can be used to explain the effects of point mutations and to shed light on the relationship between hydrophobic core architecture and coiled-coil topology.
  • Ionic stress induces fusion of mitochondria to 3-D networks: An electron
           tomography study
    • Abstract: Publication date: Available online 2 July 2018Source: Journal of Structural BiologyAuthor(s): Philip Steiner, Manja Luckner, Hubert Kerschbaum, Gerhard Wanner, Ursula Lütz-Meindl Mitochondria are central organelles for energy supply of cells and play an important role in maintenance of ionic balance. Consequently mitochondria are highly sensitive to any kind of stress to which they mainly response by disturbance of respiration, ROS production and release of cytochrome c into the cytoplasm. Many of the physiological and molecular stress reactions of mitochondria are well known, yet there is a lack of information on corresponding stress induced structural changes. 3-D visualization of high-pressure frozen cells by FIB-SEM tomography and TEM tomography as used for the present investigation provide an excellent tool for studying structure related mitochondrial stress reactions. In the present study it is shown that mitochondria in the unicellular fresh-water algal model system Micrasterias as well as in the closely related aquatic higher plant Lemna fuse to local networks as a consequence of exposure to ionic stress induced by addition of KCl, NaCl and CoCl2. In dependence on concentration and duration of the treatment, fusion of mitochondria occurs either by formation of protuberances arising from the outer mitochondrial membrane, or by direct contact of the surface of elongated mitochondria. As our results show that respiration is maintained in both model systems during ionic stress and mitochondrial fusion, as well as formation of protuberances are reversible, we assume that mitochondrial fusion is a ubiquitous process that may help the cells to cope with stress. This may occur by interconnecting the respiratory chains of the individual mitochondria and by enhancing the buffer capacity against stress induced ionic imbalance.
  • Processing apoferritin with the Appion pipeline
    • Abstract: Publication date: Available online 30 June 2018Source: Journal of Structural BiologyAuthor(s): Scott M. Stagg, Joshua H. Mendez The 3DEM map challenge provided an opportunity to test different algorithms and workflows for processing single particle cryo-EM data. We were interested in testing whether we could use the standard Appion workflow with minimal manual intervention to achieve similar or better resolution than other challengers. Another question we were interested in testing was what the influence of particle sorting and elimination would be on the resolution and quality of 3D reconstructions. Since apoferritin is historically a challenging particle for single particle reconstruction and the authors of the original map challenge data used only a fraction of the particles present in the dataset, we focused on the apoferritin dataset for our entry. We submitted a 3.7 Å map from 25,844 particles and a 3.6 Å map from 53,334 particles and after assessment were among the best of the apoferritin maps that were submitted. Here we present the details of our reconstruction strategy and compare our strategy to that of another high-scoring apoferritin map. Altogether, our results suggest that for a relatively conformationally homogeneous particle like apoferritin, including as many particles as possible after elimination of junk leads to the highest resolution, and the choice of parameters for custom mask creation can lead to subtle but significant changes in the resolution of 3D reconstructions.
  • Characterization of inter-crystallite peptides in human enamel rods
           reveals contribution by the Y allele of amelogenin
    • Abstract: Publication date: Available online 28 June 2018Source: Journal of Structural BiologyAuthor(s): Catherine Rathsam, Ramin M. Farahani, Peter G. Hains, Valentina A. Valova, Nattida Charadram, Hans Zoellner, Michael Swain, Neil Hunter Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1–6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.
  • Detection of single alpha-helices in large protein sequence sets using
           hardware acceleration
    • Abstract: Publication date: Available online 13 June 2018Source: Journal of Structural BiologyAuthor(s): Ákos Kovács, Dániel Dudola, László Nyitray, Gábor Tóth, Zoltán Nagy, Zoltán Gáspári Single alpha-helices (SAHs) are increasingly recognized as important structural and functional elements of proteins. Comprehensive identification of SAH segments in large protein datasets was largely hindered by the slow speed of the most restrictive prediction tool for their identification, FT_CHARGE on common hardware. We have previously implemented an FPGA-based version of this tool allowing fast analysis of a large number of sequences. Using this implementation, we have set up of a semi-automated pipeline capable of analyzing full UniProt releases in reasonable time and compiling monthly updates of a comprehensive database of SAH segments. Releases of this database, denoted CSAHDB, is available on the CSAHserver 2 website at An overview of human SAH-containing sequences combined with a literature survey suggests specific roles of SAH segments in proteins involved in RNA-based regulation processes as well as cytoskeletal proteins, a number of which is also linked to the development and function of synapses.
  • PSF correction in soft x-ray tomography
    • Abstract: Publication date: Available online 13 June 2018Source: Journal of Structural BiologyAuthor(s): Axel Ekman, Venera Weinhardt, Jian-Hua Chen, Gerry McDermott, Mark A. Le Gros, Carolyn Larabell In this manuscript, we introduce a linear approximation of the forward model of soft x-ray tomography (SXT), such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs.
  • High resolution crystal structure of substrate-free human neprilysin
    • Abstract: Publication date: Available online 12 June 2018Source: Journal of Structural BiologyAuthor(s): Stephen Moss, Vasanta Subramanian, K. Ravi Acharya Neprilysin is a transmembrane M13 zinc metalloprotease responsible for the degradation of several biologically active peptides including insulin, enkephalin, substance P, bradykinin, endothelin-1, neurotensin and amyloid-β. The protein has received attention for its role in modulating blood pressure responses with its inhibition producing an antihypertensive response. To date, several inhibitor bound crystal structures of the human neprilysin extracellular domain have been determined, but, a structure free of bound inhibitor or substrate has yet to be reported. Here, we report the first crystal structure free of substrate or inhibitor for the extracellular catalytic domain of human neprilysin at 1.9 Å resolution. This structure will provide a reference point for comparisons to future inhibitor or substrate bound structures. The neprilysin structure also reveals that a closed protein conformation can be adopted in protein crystals absent of bound substrate or inhibitor.
  • Structural and biochemical characterization of SpoIIIAF, a component of a
           sporulation-essential channel in Bacillus subtilis
    • Abstract: Publication date: Available online 7 June 2018Source: Journal of Structural BiologyAuthor(s): N. Zeytuni, K.A. Flanagan, L.J. Worrall, S.C. Massoni, A.H. Camp, N.C.J. Strynadka Environmental stress factors initiate the developmental process of sporulation in some Gram-positive bacteria including Bacillus subtilis. Upon sporulation initiation the bacterial cell undergoes a series of morphological transformations that result in the creation of a single dormant spore. Early in sporulation, an asymmetric cell division produces a larger mother cell and smaller forespore. Next, the mother cell septal membrane engulfs the forespore, and an essential channel, the so-called feeding-tube apparatus, is formed. This assembled channel is thought to form a transenvelope secretion complex that crosses both mother cell and forespore membranes. At least nine proteins are essential for channel formation including SpoIIQ under forespore control and the eight SpoIIIA proteins (SpoIIIAA-AH) under mother cell control. Several of these proteins share similarity with components of Gram-negative bacterial secretion systems and the flagellum. Here we report the X-ray crystallographic structure of the soluble domain of SpoIIIAF to 2.7 Å resolution. Like the channel components SpoIIIAG and SpoIIIAH, SpoIIIAF adopts a conserved ring-building motif (RBM) fold found in proteins from numerous dual membrane secretion systems of distinct function. The SpoIIIAF RBM fold contains two unique features: an extended N-terminal helix, associated with multimerization, and an insertion at a loop region that can adopt two distinct conformations. The ability of the same primary sequence to adopt different secondary structure conformations is associated with protein regulation, suggesting a dual structural and regulatory role for the SpoIIIAF RBM. We further analyzed potential interaction interfaces by structure-guided mutagenesis in vivo. Collectively, our data provide new insight into the possible roles of SpoIIIAF within the secretion-like apparatus during sporulation.
  • 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
    • 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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