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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  [3159 journals]
  • 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 BaumannTwo 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 CarazoAbstractThe 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 ZhouAbstractThe 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 ShaananProtein-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. RichardsonAbstractWe 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 PeyrinAbstractHuman 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 ElmlundAbstractCryogenic 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. AlzariAbstractThe 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. CastellinoGroup 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 KrögerAbstractThe 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 CarragherAbstractAutomated 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 SchandaAbstractThe 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 ShintakeAbstractFor 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 HeymannAbstractCryo-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 NagarAbstractSaposins 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. McKeeAbstractOsteopontin (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.
  • Structure of a prokaryotic SEFIR domain reveals two novel SEFIR-SEFIR
           interaction modes
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Hui Yang, Yun Zhu, Xing Chen, Xiaoxia Li, Sheng Ye, Rongguang ZhangAbstractSEFIR domain-containing proteins are crucial for mammalian adaptive immunity. As a unique intracellular signaling domain, the SEFIR-SEFIR interactions mediate physical protein-protein interactions in the immune signaling network, especially the IL-17- and IL-25-mediated pathways. However, due to the lack of structural information, the detailed molecular mechanism for SEFIR-SEFIR assembly remains unclear. In the present study, we solved the crystal structures of a prokaryotic SEFIR domain from Bacillus cereus F65185 (BcSEFIR), where the SEFIR domain is located at the N terminus. The structure of BcSEFIR revealed two radically distinct SEFIR-SEFIR interaction modes. In the asymmetric form, the C-terminal tail of one SEFIR binds to the helix αA and βB–αB′ segment of the other one, while in the symmetric form, the helices ηC and αE and the DE-segment compose the inter-protomer interface. The C-terminal tail of BcSEFIR, critical for asymmetric interaction, is highly conserved among the SEFIR domains of Act1 orthologs from different species, in particular three absolutely conserved residues that constitute an EXXXXPP motif. In the symmetric interaction mode, the most significant contacts made by residues on helix αE are highly conserved in Act1 SEFIR domains, constituted an RLI/LXE motif. The two novel SEFIR-SEFIR interaction modes might explain the structural basis for SEFIR domain-mediated complex assembly in signaling pathways.
  • Solution structure of an archaeal DUF61 family protein SSO0941 encoded by
           a gene in the operon of box C/D RNA protein complexes
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Tao Zhou, Xingzhe Yao, Jinfeng Wang, Yingang FengDomain of unknown function 61 (DUF61) family proteins widely exist in archaea and the genes of DUF61 proteins in crenarchaea are in an operon containing two genes of box C/D RNA protein complexes. Here we report the solution NMR structure of DUF61 family member protein SSO0941, from the hyperthermophilic archaeon Sulfolobus solfataricus. SSO0941 has a rigid core structure and flexible N- and C-terminal regions as well as a negatively-charged independent C-terminal helix. The core structure consists of N- and C-terminal subdomains, in which the C-terminal subdomain shows significant structural similarity with several nucleic acid binding proteins. The structure of SSO0941 is the first representative structure of DUF61 family proteins.Graphical abstractGraphical abstract for this article
  • Human skin barrier structure and function analyzed by cryo-EM and
           molecular dynamics simulation
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Magnus Lundborg, Ali Narangifard, Christian L. Wennberg, Erik Lindahl, Bertil Daneholt, Lars NorlénIn the present study we have analyzed the molecular structure and function of the human skin’s permeability barrier using molecular dynamics simulation validated against cryo-electron microscopy data from near native skin.The skin’s barrier capacity is located to an intercellular lipid structure embedding the cells of the superficial most layer of skin – the stratum corneum. According to the splayed bilayer model (Iwai et al., 2012) the lipid structure is organized as stacked bilayers of ceramides in a splayed chain conformation with cholesterol associated with the ceramide sphingoid moiety and free fatty acids associated with the ceramide fatty acid moiety. However, knowledge about the lipid structure’s detailed molecular organization, and the roles of its different lipid constituents, remains circumstantial.Starting from a molecular dynamics model based on the splayed bilayer model, we have, by stepwise structural and compositional modifications, arrived at a thermodynamically stable molecular dynamics model expressing simulated electron microscopy patterns matching original cryo-electron microscopy patterns from skin extremely closely. Strikingly, the closer the individual molecular dynamics models’ lipid composition was to that reported in human stratum corneum, the better was the match between the models’ simulated electron microscopy patterns and the original cryo-electron microscopy patterns. Moreover, the closest-matching model’s calculated water permeability and thermotropic behaviour were found compatible with that of human skin.The new model may facilitate more advanced physics-based skin permeability predictions of drugs and toxicants. The proposed procedure for molecular dynamics based analysis of cellular cryo-electron microscopy data might be applied to other biomolecular systems.Graphical abstractGraphical abstract for this article
  • Analytical symmetry detection in protein assemblies. I. Cyclic symmetries
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Guillaume Pagès, Elvira Kinzina, Sergei GrudininSymmetry in protein, and, more generally, in macromolecular assemblies is a key point to understand their structure, stability and function. Many symmetrical assemblies are currently present in the Protein Data Bank (PDB) and some of them are among the largest solved structures, thus an efficient computational method is needed for the exhaustive analysis of these. The cyclic symmetry groups represent the most common assemblies in the PDB. These are also the building blocks for higher-order symmetries. This paper presents a mathematical formulation to find the position and the orientation of the symmetry axis in a cyclic symmetrical protein assembly, and also to assess the quality of this symmetry. Our method can also detect symmetries in partial assemblies.We provide an efficient C++ implementation of the method and demonstrate its efficiency on several examples including partial assemblies and pseudo symmetries. We also compare the method with two other published techniques and show that it is significantly faster on all the tested examples. Our method produces results with a machine precision, its cost function is solely based on 3D Euclidean geometry, and most of the operations are performed analytically. The method is available at The graphical user interface of the method built for the SAMSON platform is available at abstractGraphical abstract for this article
  • Substrate-bound structures of a ketoreductase from amphotericin modular
           polyketide synthase
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Chenguang Liu, Meijuan Yuan, Xu Xu, Lei Wang, Adrian T. Keatinge-Clay, Zixin Deng, Shuangjun Lin, Jianting ZhengKetoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereochemical control of PKS KR domains.Graphical abstractGraphical abstract for this article
  • Image processing techniques for high-resolution structure determination
           from badly ordered 2D crystals
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Nikhil Biyani, Sebastian Scherer, Ricardo D. Righetto, Julia Kowal, Mohamed Chami, Henning StahlbergAbstract2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10–12 Å can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5 Å.
  • Structural and functional analysis of Erwinia amylovora SrlD. The first
           crystal structure of a sorbitol-6-phosphate 2-dehydrogenase
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Marco Salomone-Stagni, Joseph D. Bartho, Eeshan Kalita, Martin Rejzek, Robert A. Field, Dom Bellini, Martin A. Walsh, Stefano BeniniAbstractSorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved.Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
  • A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for
           nuclear export
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Norman R. Watts, Elif Eren, Xiaolei Zhuang, Yun-Xing Wang, Alasdair C. Steven, Paul T. WingfieldAbstractHIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.
  • Delivery of femtolitre droplets using surface acoustic wave based
           atomisation for cryo-EM grid preparation
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Dariush Ashtiani, Hari Venugopal, Matthew Belousoff, Bradley Spicer, Johnson Mak, Adrian Neild, Alex de MarcoAbstractCryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6–8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.
  • Blind estimation of DED camera gain in Electron Microscopy
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): C.O.S. Sorzano, E. Fernández-Giménez, V. Peredo-Robinson, J. Vargas, T. Majtner, G. Caffarena, J. Otón, J.L. Vilas, J.M. de la Rosa-Trevín, R. Melero, J. Gómez-Blanco, J. Cuenca, L. del Cano, P. Conesa, R. Marabini, J.M. CarazoAbstractThe introduction of Direct Electron Detector (DED) videos in the Electron Microscope field has boosted Single Particle Analysis to a point in which it is currently considered to be a key technique in Structural Biology. In this article we introduce an approach to estimate the DED camera gain at each pixel from the movies themselves. This gain is needed to have the set of recorded frames into a coherent gray level range, homogeneous over the whole image. The algorithm does not need any other input than the DED movie itself, being capable of providing an estimate of the camera gain image, helping to identify dead pixels and cases of incorrectly calibrated cameras. We propose the algorithm to be used either to validate the experimentally acquired gain image (for instance, to follow its possible change over time) or to verify that there is no residual gain image after experimentally correcting for the camera gain. We show results for a number of DED camera models currently in use (DE, Falcon II, Falcon 3, and K2).
  • New zonal structure and transition of the membrane to mammillae in the
           eggshell of chicken Gallus domesticus
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Yunong Li, Yang Li, Shirong Liu, Yang Tang, Bing Mo, Hui LiaoAbstractAvian eggshell is a typical bio-engineered ceramics characterized by layer structures. These layers are categorized mainly by the form of crystalline calcite. Whether there exist other layer structures, how the membrane layer is transformed to the carbonate one, what form the carbonate takes after the transition. These questions remain to be clarified. Here we examine the eggshell of chicken Gallus domesticus by optical microscope, scanning electron microscope and transmission electron microscope. We find that there exists another layer structure defined by variation of organic matrices. The transition from the membrane to the mammillary cones is implemented through the calcium reserve assemblies or the mammillary cores. The integrity of the transitional structure was weakens as the reserved calcium is displaced, and loses completely in about 10 days of incubation. As the first deposited carbonate layer after the transition, the mammillary cones comprise amorphous calcium carbonate and clusters/assemblies of calcite crystallites the size about a nanometer, plus bubble pores extending preferentially in the lateral direction. Our results provide new insights into the structure and component of the avian eggshell, and may help decipher the constitution of the bio-ceramics in the perspective of material science.
  • Baculovirus-driven protein expression in insect cells: A benchmarking
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Peggy Stolt-Bergner, Christian Benda, Tim Bergbrede, Hüseyin Besir, Patrick H.N. Celie, Cindy Chang, David Drechsel, Ariane Fischer, Arie Geerlof, Barbara Giabbai, Joop van den Heuvel, Georg Huber, Wolfgang Knecht, Anita Lehner, Regis Lemaitre, Kristina Nordén, Gwynn Pardee, Ines Racke, Kim Remans, Astrid SanderAbstractBaculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
  • Structure of the DNA-binding domain of human myelin-gene regulatory factor
           reveals its potential protein-DNA recognition mode
    • Abstract: Publication date: August 2018Source: Journal of Structural Biology, Volume 203, Issue 2Author(s): Baohua Chen, Yun Zhu, Sheng Ye, Rongguang ZhangAbstractMyelin-gene regulatory factor (MYRF) is a membrane-bound transcription factors, which is responsible for the differentiation of oligodendrocytes and myelination of central nervous system. Followed by a self-cleavage by the intramolecular chaperone auto-processing (ICA) domain, DNA-binding domain (DBD) of MYRF is released from the endoplasmic reticulum (ER) and was then translocated to the nucleus to regulate gene expression. In present work, we have solved the crystal structure of the human MYRF-DBD to 1.85-Å resolution. It exhibits a typical s-type Ig-fold and packs as symmetric trimeric form in the crystal via hydrogen-bond networks in three regions. Accordingly, we identified a couple of key residues on MYRF-DBD, which might play important roles in DNA-binding, in particular Arg521 on its C-terminal tail. The R521A mutant of DBD showed only 17% affinity to dsDNA targets compared to wild-type DBD. Then we built a plausible protein-DNA binding model of MYRF-DBD, which will help to elucidate its mechanism in DNA-binding and transcriptional regulation.
  • 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 ChiuAbstractAn 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 ChengAbstractGraphene 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. StrelkovAbstractα-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 HeymannAbstractThe 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. PotterAbstractRecent 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 OhfujiAbstractCross-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-HorkawiczAbstractIn 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-MeindlAbstractMitochondria 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. MendezAbstractThe 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 HunterAbstractProteins 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.
  • Analytical symmetry detection in protein assemblies. II. Dihedral and
           cubic symmetries
    • Abstract: Publication date: Available online 15 June 2018Source: Journal of Structural BiologyAuthor(s): Guillaume Pagès, Sergei GrudininProtein assemblies are often symmetric, as this organization has many advantages compared to individual proteins. Complex protein structures thus very often possess high-order symmetries. Detection and analysis of these symmetries has been a challenging problem and no efficient algorithms have been developed so far. This paper presents the extension of our cyclic symmetry detection method for higher-order symmetries with multiple symmetry axes. These include dihedral and cubic, i.e., tetrahedral, octahedral, and icosahedral, groups. Our method assesses the quality of a particular symmetry group and also determines all of its symmetry axes with a machine precision. The method comprises discrete and continuous optimization steps and is applicable to assemblies with multiple chains in the asymmetric subunits or to those with pseudo-symmetry.We implemented the method in C++ and exhaustively tested it on all 51,358 symmetric assemblies from the Protein Data Bank (PDB). It allowed us to study structural organization of symmetric assemblies solved by X-ray crystallography, and also to assess the symmetry annotation in the PDB. For example, in 1.6% of the cases we detected a higher symmetry group compared to the PDB annotation, and we also detected several cases with incorrect annotation. The method is available at The graphical user interface of the method built for the SAMSON platform is available at abstractGraphical abstract for this article
  • 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 GáspáriAbstractSingle 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 LarabellAbstractIn 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 AcharyaAbstractNeprilysin 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. StrynadkaAbstractEnvironmental 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.
  • Identification of a novel tetrameric structure for human apolipoprotein-D
    • Abstract: Publication date: Available online 6 June 2018Source: Journal of Structural BiologyAuthor(s): Claudia S. Kielkopf, Jason K.K. Low, Yee-Foong Mok, Surabhi Bhatia, Tony Palasovski, Aaron J. Oakley, Andrew E. Whitten, Brett Garner, Simon H.J. BrownApolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer’s disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer’s disease mouse models.Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.Graphical abstractGraphical abstract for this article
  • Reductive power of the archaea right-handed coiled coil nanotube (RHCC-NT)
           and incorporation of mercury clusters inside protein cages
    • Abstract: Publication date: Available online 5 June 2018Source: Journal of Structural BiologyAuthor(s): Matthew McDougall, Kevin McEleney, Olga Francisco, Benchmen Trieu, Efehi Kelly Ogbomo, Gregg Tomy, Jörg StetefeldAbstractCoiled coils are well described as powerful oligomerization motifs and exhibit a large diversity of functions, including gene regulation, cell division, membrane fusion and drug extrusion. The archaea S-layer originated right-handed coiled coil –RHCC-NT- is characterized by extreme stability and is free of cysteine and histidine moieties. In the current study, we have followed a multidisciplinary approach to investigate the capacity of RHCC-NT to bind a variety of ionic complex metal ions. At the outside of the RHCC-NT, one mercury ion forms an electrostatic interaction with the S-methyl moiety of the single methionine residue present in each coil. We demonstrate that RHCC-NT is reducing and incorporating metallic mercury in the large-sized interior cavities which are lined up along the tetrameric channel.
  • 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 GlaubitzAbstractKrokinobacter 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.
  • cryoem-cloud-tools: A software platform to deploy and manage cryo-EM jobs
           in the cloud
    • Abstract: Publication date: Available online 1 June 2018Source: Journal of Structural BiologyAuthor(s): Michael A. Cianfrocco, Indrajit Lahiri, Frank DiMaio, Andres E. LeschzinerAbstractAccess to streamlined computational resources remains a significant bottleneck for new users of cryo-electron microscopy (cryo-EM). To address this, we have developed tools that will submit cryo-EM analysis routines and atomic model building jobs directly to Amazon Web Services (AWS) from a local computer or laptop. These new software tools (“cryoem-cloud-tools”) have incorporated optimal data movement, security, and cost-saving strategies, giving novice users access to complex cryo-EM data processing pipelines. Integrating these tools into the RELION processing pipeline and graphical user interface we determined a 2.2 Å structure of ß-galactosidase in ∼55 h on AWS. We implemented a similar strategy to submit Rosetta atomic model building and refinement to AWS. These software tools dramatically reduce the barrier for entry of new users to cloud computing for cryo-EM and are freely available at
  • 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 CegelskiAbstractWhole-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.
  • Plasmin-driven fibrinolysis in a quasi-two-dimensional nanoscale fibrin
    • Abstract: Publication date: Available online 30 May 2018Source: Journal of Structural BiologyAuthor(s): Tímea Feller, Jolán Hársfalvi, Csilla Csányi, Balázs Kiss, Miklós KellermayerAbstractFibrin plays a fundamentally important role during hemostasis. To withstand the shear forces of blood flow and prevent embolisation, fibrin monomers form a three-dimensional polymer network that serves as an elastic scaffold for the blood clot. The complex spatial hierarchy of the fibrin meshwork, however, severely complicates the exploration of structural features, mechanical properties and molecular changes associated with the individual fibers of the clot. Here we developed a quasi-two-dimensional nanoscale fibrin matrix that enables the investigation of fibrin properties by topographical analysis using atomic force microscopy. The average thickness of the matrix was ∼50 nm, and structural features of component fibers were accessible. The matrix could be lysed with plasmin following rehydration. By following the topology of the matrix during lysis, we were able to uncover the molecular mechanisms of the process. Fibers became flexible but retained axial continuity for an extended time period, indicating that lateral interactions between protofibrils are disrupted first, but the axial interactions remain stable. Nearby fibers often fused into bundles, pointing at the presence of a cohesional force between them. Axial fiber fragmentation rapidly took place in the final step. Conceivably, the persisting axial integrity and cohesion of the fibrils assist to maintain global clot structure, to prevent microembolism, and to generate a high local plasmin concentration for the rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed and tested here provides a unique insight into the molecular mechanisms behind the structural and mechanical features of fibrin and its proteolytic degradation.
  • Biochemical characterization of the skeletal matrix of the massive coral,
           Porites australiensis – The saccharide moieties and their localization
    • Abstract: Publication date: Available online 30 May 2018Source: Journal of Structural BiologyAuthor(s): Takeshi Takeuchi, Laurent Plasseraud, Isabelle Ziegler-Devin, Nicolas Brosse, Chuya Shinzato, Noriyuki Satoh, Frédéric MarinAbstractTo construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.
  • The protofilament architecture of a de novo designed coiled coil-based
           amyloidogenic peptide
    • Abstract: Publication date: Available online 29 May 2018Source: Journal of Structural BiologyAuthor(s): Mônica Santos de Freitas, Raheleh Rezaei Araghi, Enrico Brandenburg, Jork Leiterer, Franziska Emmerling, Kristin Folmert, Ulla I.M. Gerling-Driessen, Benjamin Bardiaux, Christoph Böttcher, Kevin Pagel, Anne Diehl, Hans v. Berlepsch, Hartmut Oschkinat, Beate KokschAbstractAmyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson’s and Alzheimer’s diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements.
  • Mechanical variations in proteins with large-scale motions highlight the
           formation of structural locks
    • Abstract: Publication date: Available online 28 May 2018Source: Journal of Structural BiologyAuthor(s): Sophie Sacquin-MoraAbstractProtein function depends just as much on flexibility as on structure, and in numerous cases, a protein’s biological activity involves transitions that will impact both its conformation and its mechanical properties. Here, we use a coarse-grain approach to investigate the impact of structural changes on protein flexibility. More particularly, we focus our study on proteins presenting large-scale motions. We show how calculating directional force constants within residue pairs, and investigating their variation upon protein closure, can lead to the detection of a limited set of residues that form a structural lock in the protein’s closed conformation. This lock, which is composed of residues whose side-chains are tightly interacting, highlights a new class of residues that are important for protein function by stabilizing the closed structure, and that cannot be detected using earlier tools like local rigidity profiles or distance variations maps, or alternative bioinformatics approaches, such as coevolution scores.
  • Atomic structure of a rationally engineered gene delivery vector, AAV2.5
    • Abstract: Publication date: Available online 18 May 2018Source: Journal of Structural BiologyAuthor(s): Matthew Burg, Claire Rosebrough, Lauren M. Drouin, Antonette Bennett, Mario Mietzsch, Paul Chipman, Robert McKenna, Duncan Sousa, Mark Potter, Barry Byrne, R. Jude Samulski, Mavis Agbandje-McKennaAbstractAAV2.5 represents the first structure-guided in-silico designed Adeno-associated virus (AAV) gene delivery vector. This engineered vector combined the receptor attachment properties of AAV serotype 2 (AAV2) with the muscle tropic properties of AAV1, and exhibited an antibody escape phenotype because of a modified antigenic epitope. To confirm the design, the structure of the vector was determined to a resolution of 2.78 Å using cryo-electron microscopy and image reconstruction. The structure of the major viral protein (VP), VP3, was ordered from residue 219 to 736, as reported for other AAV structures, and the five AAV2.5 residues exchanged from AAV2 to AAV1, Q263A, T265 (insertion), N706A, V709A, and T717N, were readily interpretable. Significantly, the surface loops containing these residues adopt the AAV1 conformation indicating the importance of amino acid residues in dictating VP structure.
  • Effects of flexibility of the α2 chain of type I collagen on
           collagenase cleavage
    • Abstract: Publication date: Available online 12 May 2018Source: Journal of Structural BiologyAuthor(s): Arya Mekkat, Erik Poppleton, Bo An, Robert Visse, Hideaki Nagase, David L. Kaplan, Barbara Brodsky, Yu-Shan LinAbstractCleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.
  • Collagen Gly missense mutations: Effect of residue identity on collagen
           structure and integrin binding
    • Abstract: Publication date: Available online 11 May 2018Source: Journal of Structural BiologyAuthor(s): Yimin Qiu, Arya Mekkat, Hongtao Yu, Sezin Yigit, Samir Hamaia, Richard W. Farndale, David L. Kaplan, Yu-Shan Lin, Barbara BrodskyAbstractGly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.
  • 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. JaroniecAbstractThe 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 LangeAbstractIntra-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 LuAbstractHigh-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 HongAbstractMany 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 HabensteinAbstractREMORINs 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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Heriot-Watt University
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