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Cryo-EM

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https://www.readbyqxmd.com/read/28191458/structural-study-of-heterogeneous-biological-samples-by-cryoelectron-microscopy-and-image-processing
#1
REVIEW
H E White, A Ignatiou, D K Clare, E V Orlova
In living organisms, biological macromolecules are intrinsically flexible and naturally exist in multiple conformations. Modern electron microscopy, especially at liquid nitrogen temperatures (cryo-EM), is able to visualise biocomplexes in nearly native conditions and in multiple conformational states. The advances made during the last decade in electronic technology and software development have led to the revelation of structural variations in complexes and also improved the resolution of EM structures. Nowadays, structural studies based on single particle analysis (SPA) suggests several approaches for the separation of different conformational states and therefore disclosure of the mechanisms for functioning of complexes...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28188547/structurally-conserved-channels-in-cyanobacterial-and-plant-photosystem-ii
#2
Naoki Sakashita, Hiroshi C Watanabe, Takuya Ikeda, Hiroshi Ishikita
In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn4Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn4Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel)...
February 10, 2017: Photosynthesis Research
https://www.readbyqxmd.com/read/28183995/structure-of-a-eukaryotic-voltage-gated-sodium-channel-at-near-atomic-resolution
#3
Huaizong Shen, Qiang Zhou, Xiaojing Pan, Zhangqiang Li, Jianping Wu, Nieng Yan
Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryo-EM structure of a putative Nav channel from American cockroach (designated NavPaS) at 3.8-Å resolution. The voltage sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops...
February 9, 2017: Science
https://www.readbyqxmd.com/read/28178455/seeing-hpv-in-the-new-light-offers-a-glimpse-of-heparin
#4
Peter C Angeletti
In this issue of Structure, Guan et al. (2017) describe cryo-EM mapping of human papillomavirus 16 (HPV16) capsids that propose locations for L2 and heparin binding sites. The high resolution of the modern cryo-EM methods (in this case down to 4.3 Å) opens great opportunities to probe conformational changes that take place during virus-receptor binding.
February 7, 2017: Structure
https://www.readbyqxmd.com/read/28176812/quantification-of-detergents-complexed-with-membrane-proteins
#5
Vincent Chaptal, Frédéric Delolme, Arnaud Kilburg, Sandrine Magnard, Cédric Montigny, Martin Picard, Charlène Prier, Luca Monticelli, Olivier Bornert, Morgane Agez, Stéphanie Ravaud, Cédric Orelle, Renaud Wagner, Anass Jawhari, Isabelle Broutin, Eva Pebay-Peyroula, Jean-Michel Jault, H Ronald Kaback, Marc le Maire, Pierre Falson
Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins...
February 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28169832/interface-between-40s-exit-channel-protein-us7-rps5-and-eif2%C3%AE-modulates-start-codon-recognition-in-vivo
#6
Jyothsna Visweswaraiah, Alan G Hinnebusch
The eukaryotic pre-initiation complex (PIC) bearing the eIF2•GTP•Met-tRNAi(Met) ternary complex (TC) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes rearrangement of the PIC from an open, scanning to a closed, arrested conformation. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interface between 40S protein Rps5/uS7 and eIF2α between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2α contacts favored in the open complex increase initiation at suboptimal sites, and uS7-S223D stabilizes TC binding to PICs reconstituted with a UUG start codon, indicating inappropriate rearrangement to the closed state...
February 7, 2017: ELife
https://www.readbyqxmd.com/read/28165473/cryosparc-algorithms-for-rapid-unsupervised-cryo-em-structure-determination
#7
Ali Punjani, John L Rubinstein, David J Fleet, Marcus A Brubaker
Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer...
February 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/28164115/modeling-beta-traces-for-beta-barrels-from-cryo-em-density-maps
#8
Dong Si, Jing He
Cryo-electron microscopy (cryo-EM) has produced density maps of various resolutions. Although α-helices can be detected from density maps at 5-8 Å resolutions, β-strands are challenging to detect at such density maps due to close-spacing of β-strands. The variety of shapes of β-sheets adds the complexity of β-strands detection from density maps. We propose a new approach to model traces of β-strands for β-barrel density regions that are extracted from cryo-EM density maps. In the test containing eight β-barrels extracted from experimental cryo-EM density maps at 5...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28148783/beta-propiolactone-inactivation-of-coxsackievirus-a16-induces-structural-alteration-and-surface-modification-of-viral-capsids
#9
Chen Fan, Xiaohua Ye, Zhiqiang Ku, Liangliang Kong, Qingwei Liu, Cong Xu, Yao Cong, Zhong Huang
: Beta-propiolactone (BPL) is an inactivating agent widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we presented cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N-terminus of VP1 capsid protein as well as the absence of pocket factor...
February 1, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28143931/mechanistic-insights-into-caspase-9-activation-by-the-structure-of-the-apoptosome-holoenzyme
#10
Yini Li, Mengying Zhou, Qi Hu, Xiao-Chen Bai, Weiyun Huang, Sjors H W Scheres, Yigong Shi
Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub...
January 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28138066/the-translation-elongation-cycle-capturing-multiple-states-by-cryo-electron-microscopy
#11
REVIEW
Joachim Frank
During the work cycle of elongation, the ribosome, a molecular machine of vast complexity, exists in a large number of states distinguished by constellation of its subunits, its subunit domains and binding partners. Single-particle cryogenic electron microscopy (cryo-EM), developed over the past 40 years, is uniquely suited to determine the structure of molecular machines in their native states. With the emergence, 10 years ago, of unsupervised clustering techniques in the analysis of single-particle data, it has been possible to determine multiple structures from a sample containing ribosomes equilibrating in different thermally accessible states...
March 19, 2017: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
https://www.readbyqxmd.com/read/28138065/dynamics-of-ires-mediated-translation
#12
REVIEW
Alex G Johnson, Rosslyn Grosely, Alexey N Petrov, Joseph D Puglisi
Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning...
March 19, 2017: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
https://www.readbyqxmd.com/read/28133813/the-hiv-1-envelope-glycoprotein-structure-nailing-down-a-moving-target
#13
REVIEW
Andrew B Ward, Ian A Wilson
Structure determination of the HIV-1 envelope glycoprotein (Env) presented a number of challenges, but several high-resolution structures have now become available. In 2013, cryo-EM and x-ray structures of soluble, cleaved SOSIP Env trimers from the clade A BG505 strain provided the first glimpses into the Env trimer fold as well as more the variable regions. A recent cryo-EM structure of a native full-length trimer without any stabilizing mutations had the same core structure, but revealed new insights and features...
January 2017: Immunological Reviews
https://www.readbyqxmd.com/read/28129535/cryo-em-captures-the-dynamics-of-ion-channel-opening
#14
John L Rubinstein
In this issue of Cell, Hite and MacKinnon (2017) report the open conformation structure of Slo2.2, a neuronal Na(+)-activated K(+) channel. More importantly, 3D classification of electron cryomicroscopy (cryo-EM) images allows visualization of the structural transition that occurs as the open probability of individual channels increases with added sodium.
January 26, 2017: Cell
https://www.readbyqxmd.com/read/28115689/structural-insights-into-the-functional-cycle-of-the-atpase-module-of-the-26s-proteasome
#15
Marc Wehmer, Till Rudack, Florian Beck, Antje Aufderheide, Günter Pfeifer, Jürgen M Plitzko, Friedrich Förster, Klaus Schulten, Wolfgang Baumeister, Eri Sakata
In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA(+) ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4)...
February 7, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28115258/cryo-em-analysis-of-a-domain-antibody-bound-rotary-atpase-complex
#16
Roberta B Davies, Callum Smits, Andrew Sw Wong, Daniela Stock, Mary Christie, Sara Sandin, Alastair G Stewart
The bacterial A/V-type ATPase/synthase rotary motor couples ATP hydrolysis/synthesis with proton translocation across biological membranes. The A/V-type ATPase/synthase from Thermus thermophilus has been extensively studied both structurally and functionally for many years. Here we provide an 8.7 Å resolution cryo-electron microscopy 3D reconstruction of this complex bound to single-domain antibody fragments, small monomeric antibodies containing just the variable heavy domain. Docking of known structures into the density revealed the molecular orientation of the domain antibodies, suggesting that structure determination of co-domain antibody:protein complexes could be a useful avenue for unstable or smaller proteins...
January 20, 2017: Journal of Structural Biology
https://www.readbyqxmd.com/read/28112732/structural-snapshot-of-cytoplasmic-pre-60s-ribosomal-particles-bound-by-nmd3-lsg1-tif6-and-reh1
#17
Chengying Ma, Shan Wu, Ningning Li, Yan Chen, Kaige Yan, Zhifei Li, Lvqin Zheng, Jianlin Lei, John L Woolford, Ning Gao
A key step in ribosome biogenesis is the nuclear export of pre-ribosomal particles. Nmd3, a highly conserved protein in eukaryotes, is a specific adaptor required for the export of pre-60S particles. Here we used cryo-electron microscopy (cryo-EM) to characterize Saccharomyces cerevisiae pre-60S particles purified with epitope-tagged Nmd3. Our structural analysis indicates that these particles belong to a specific late stage of cytoplasmic pre-60S maturation in which ribosomal proteins uL16, uL10, uL11, eL40 and eL41 are deficient, but ribosome assembly factors Nmd3, Lsg1, Tif6 and Reh1 are present...
January 23, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28112730/cotranslational-folding-of-spectrin-domains-via-partially-structured-states
#18
Ola B Nilsson, Adrian A Nickson, Jeffrey J Hollins, Stephan Wickles, Annette Steward, Roland Beckmann, Gunnar von Heijne, Jane Clarke
How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes...
January 23, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28112729/structural-basis-of-dual-ca-2-ph-regulation-of-the-endolysosomal-trpml1-channel
#19
Minghui Li, Wei K Zhang, Nicole M Benvin, Xiaoyuan Zhou, Deyuan Su, Huan Li, Shu Wang, Ioannis E Michailidis, Liang Tong, Xueming Li, Jian Yang
The activities of organellar ion channels are often regulated by Ca(2+) and H(+), which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca(2+)/pH regulation of TRPML1, a Ca(2+)-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations...
January 23, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28109880/dynamics-of-the-eag1-k-channel-selectivity-filter-assessed-by-molecular-dynamics-simulations
#20
Harald Bernsteiner, Michael Bründl, Anna Stary-Weinzinger
EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4...
January 19, 2017: Biochemical and Biophysical Research Communications
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