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Journal of Structural Biology

Cyril F Reboul, Simon Kiesewetter, Michael Eager, Matthew Belousoff, Tiangang Cui, Hans De Sterck, Dominika Elmlund, Hans Elmlund
Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction...
August 6, 2018: Journal of Structural Biology
Yuhang Wang, Mrinal Shekhar, Darren Thifault, Christopher J Williams, Ryan Mcgreevy, Jane Richardson, Abhishek Singharoy, Emad Tajkhorshid
Accurate structure determination from electron density maps at 3-5 Å resolution necessitates a balance between extensive global and local sampling of atomistic models, yet with stereochemical correctness of backbone and sidechain geometries. Molecular Dynamics Flexible Fitting (MDFF), particularly through a resolution-exchange scheme, ReMDFF, provides a robust way of achieving this balance for hybrid structure determination. Employing two high-resolution density maps, namely that of β-galactosidase at 3.2 Å and TRPV1 at 3...
August 6, 2018: Journal of Structural Biology
Ariel E Mechaly, Ahmed Haouz, Nathalie Sassoon, Alejandro Buschiazzo, Jean-Michel Betton, Pedro M Alzari
The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones...
August 4, 2018: Journal of Structural Biology
Genki Terashi, Daisuke Kihara
Protein tertiary structure modeling is a critical step for the interpretation of three dimensional (3D) election microscopy density.Our group participated the 2015/2016 EM Model Challenge using the MAINMAST software for a de novo main chain modeling.The software generates local density points using the mean shifting algorithm, and connects them into Cα models by calculating the minimum spanning tree and the longest path. Subsequently, full atom structure models are generated, which are subject to structural refinement...
July 31, 2018: Journal of Structural Biology
Cunjia Qiu, Yue Yuan, Jaroslav Zajicek, Zhong Liang, Rashna D Balsara, Teresa Brito-Robionson, Shaun W Lee, Victoria A Ploplis, Francis J Castellino
Group A Streptococcus pyogenes (GAS) is a causative agent of pharyngeal and dermal infections in humans. A major virulence determinant of GAS is its dimeric signature fibrillar M-protein (M-Prt), which is evolutionarily designed in modules, ranging from a hypervariable extracellular N-terminal region to a progressively more highly conserved C-terminus that is covalently anchored to the cell wall. Of the >250 GAS isolates classified, only the subset of skin-trophic Pattern D strains expresses a specific serotype of M-Prt, PAM, that directly binds to host human plasminogen (hPg) via its extracellular NH2 -terminal variable A-domain region...
July 30, 2018: Journal of Structural Biology
Thomas C Terwilliger, Paul D Adams, Pavel V Afonine, Oleg V Sobolev
A recently-developed method for identifying a compact, contiguous region representing the unique part of a density map was applied to 218 cryo-EM maps with resolutions of 4.5 Å or better. The key elements of the segmentation procedure are (1) identification of all regions of density above a threshold and (2) choice of a unique set of these regions, taking symmetry into consideration, that maximize connectivity and compactness. This segmentation approach was then combined with tools for automated map sharpening and model-building to generate models for the 12 maps in the 2016 cryo-EM model challenge in a fully automated manner...
July 28, 2018: Journal of Structural Biology
Damian Pawolski, Christoph Heintze, Ingo Mey, Claudia Steinem, Nils Kröger
The genetically-controlled formation of complex-shaped inorganic materials by living organisms is an intriguing phenomenon. It illustrates our incomplete understanding of biological morphogenesis and demonstrates the feasibility of ecologically benign routes for materials technology. Amorphous SiO2 (silica) is taxonomically the most widespread biomineral, with diatoms, a large group of single-celled microalgae, being the most prolific producers. Silica is the main component of diatom cell walls, which exhibit species-specific patterns of pores that are hierarchically arranged and endow the material with advantageous properties...
July 28, 2018: Journal of Structural Biology
Anchi Cheng, Edward T Eng, Lambertus Alink, William J Rice, Kelsey D Jordan, Laura Y Kim, Clinton S Potter, Bridget Carragher
Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (+/- 0...
July 25, 2018: Journal of Structural Biology
Slavica Jonić
This article presents a methodology to assess a set of density maps, as used in the Blind Assessment Phase of the 2015/2016 Map Challenge (EMDataBank Validation Challenges). The synthetic and experimental cryo-electron microscopy (cryo-EM) density maps obtained by different single particle analysis protocols and by different participants, submitted in the Challenge Phase for assessment, were analyzed with this methodology and the obtained results are presented and discussed here. The goal of using such a methodology was to blindly identify the density maps with globally similar structural information, meaning the maps with the structural information mostly "reproduced" by different protocols...
July 20, 2018: Journal of Structural Biology
Catherine Bougault, Isabel Ayala, Waldemar Vollmer, Jean-Pierre Simorre, Paul Schanda
The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, 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...
July 19, 2018: Journal of Structural Biology
Martin Cheung, Hidehito Adaniya, Cathal Cassidy, Masao Yamashita, Kun-Lung Li, Seita Taba, Tsumoru Shintake
For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches...
July 18, 2018: Journal of Structural Biology
J Bernard Heymann
Cryo-electron microscopy (cryoEM) is capable of achieving near-atomic resolution of biomolecular structures due to recent advances in hardware. Despite the long history of image processing software development for cryoEM, uncertainty about best practices and validation remains. The Map Challenge was therefore designed to test the current state of single particle reconstruction. As the first such challenge, the participants were given the freedom to analyze the cases in whichever way they wanted. Therefore, the maps submitted feature different sizes, sampling and orientations, making assessment non-trivial...
July 17, 2018: Journal of Structural Biology
Ahmad Gebai, Alexei Gorelik, Bhushan Nagar
Saposins are accessory proteins that aid in the degradation of sphingolipids by hydrolytic enzymes. Their structure usually comprises four α-helices arranged in various conformations including an open, V-shaped form that is generally associated with the ability to interact with membranes and/or enzymes to accentuate activity. Saposin D is required by the lysosomal hydrolase, acid ceramidase, which breaks down ceramide into sphingosine and free fatty acid, to display optimal activity. The structure of saposin D was previously determined in an inactive conformation, revealing a monomeric, closed and compact form...
July 17, 2018: Journal of Structural Biology
Yung-Ching Chien, Ahmad Mansouri, Wenge Jiang, Saeed R Khan, Jeffrey J Gray, Marc D McKee
Osteopontin (OPN) is a significant component of kidney stone matrix and a key modulator of stone formation. Here, we investigated the effects of different phosphorylated states of a synthesized peptide of OPN (the ASARM peptide; acidic, serine- and aspartate-rich motif) on calcium oxalate dihydrate (COD) crystals, a major mineral phase of kidney stones. In vitro, phosphorylated OPN-ASARM peptides strongly inhibited COD crystal growth in solution as compared to the nonphosphorylated state, with increasing inhibitory potency correlating with the degree of peptide phosphorylation...
July 16, 2018: Journal of Structural Biology
Andriy Kryshtafovych, Paul D Adams, Catherine L Lawson, Wah Chiu
An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail...
July 12, 2018: Journal of Structural Biology
Eugene Palovcak, Feng Wang, Shawn Q Zheng, Zanlin Yu, Sam Li, Miguel Betegon, David Bulkley, David A Agard, Yifan Cheng
Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface...
July 11, 2018: Journal of Structural Biology
Dmytro Guzenko, Sergei V Strelkov
α-Helical coiled coils (CCs) represent an important, highly regular protein folding motif. To date, many thousands of CC structures have been determined experimentally. Their geometry is usually modelled by theoretical equations introduced by F. Crick that involve a predefined set of parameters. Here we have addressed the problem of efficient CC parameterization from scratch by performing a statistical evaluation of all available CC structures. The procedure is based on the principal component analysis and yields a minimal set of independent parameters that provide for the reconstruction of the complete CC structure at a required precision...
July 9, 2018: Journal of Structural Biology
J Bernard Heymann
The Bsoft package is aimed at processing electron micrographs for the determination of the three-dimensional structures of biological specimens. Recent advances in hardware allow us to solve structures to near atomic resolution using single particle analysis (SPA). The Map Challenge offered me an opportunity to test the ability of Bsoft to produce reconstructions from cryo-electron micrographs at the best resolution. I also wanted to understand what needed to be done to work towards full automation with validation...
July 5, 2018: Journal of Structural Biology
William J Rice, Anchi Cheng, Alex J Noble, Edward T Eng, Laura Y Kim, Bridget Carragher, Clinton S Potter
Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected...
July 4, 2018: Journal of Structural Biology
Krzysztof Szczepaniak, Jan Ludwiczak, Aleksander Winski, Stanislaw Dunin-Horkawicz
In protein modelling and design, an understanding of the relationship between sequence and structure is essential. Using parallel, homotetrameric coiled-coil structures as a model system, we demonstrated that machine learning techniques can be used to predict structural parameters directly from the sequence. Coiled coils are regular protein structures, which are of great interest as building blocks for assembling larger nanostructures. They are composed of two or more alpha-helices wrapped around each other to form a supercoiled bundle...
July 2, 2018: Journal of Structural Biology
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