Journal articles: 'Cryo EM 2'

1

Fujiyoshi, Yoshinori. "Drug Rescuing by Cryo-EM." Proceedings for Annual Meeting of The Japanese Pharmacological Society WCP2018 (2018): SY16–2. http://dx.doi.org/10.1254/jpssuppl.wcp2018.0_sy16-2.

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Chen, Xizi, Mengjie Liu, Yuan Tian, Jiabei Li, Yilun Qi, Dan Zhao, Zihan Wu, et al. "Cryo-EM structure of human mTOR complex 2." Cell Research 28, no. 5 (March 22, 2018): 518–28. http://dx.doi.org/10.1038/s41422-018-0029-3.

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Henderson, Richard, and Samar Hasnain. "`Cryo-EM': electron cryomicroscopy, cryo electron microscopy or something else?" IUCrJ 10, no. 5 (September 1, 2023): 519–20. http://dx.doi.org/10.1107/s2052252523006759.

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Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. During the last ten years, cryoEM has become widely available and has provided a major boost to structure determination of membrane proteins and large multi-protein complexes. Many of the structures have now been made available at resolutions around 2 Å, where fundamental questions regarding enzyme mechanisms can be addressed. Over the years, the abbreviation cryoEM has been understood to stand for different things. We wish the wider community to engage and clarify the definition of cryoEM so that the expanding literature involving cryoEM is unified.

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Sherman, M. B., F. Nasar, and S. C. Weaver. "Cryo-EM Reconstruction Of Eilat Alphavirus." Microscopy and Microanalysis 18, S2 (July 2012): 74–75. http://dx.doi.org/10.1017/s143192761200222x.

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Kamyshinsky, Roman, Yury Chesnokov, Liubov Dadinova, Andrey Mozhaev, Alexander Vasiliev, and Eleonora Shtykova. "Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S7. http://dx.doi.org/10.21103/ijbm.11.suppl_1.or2.

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Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.

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Mei, Kunrong, Yan Li, Shaoxiao Wang, Guangcan Shao, Jia Wang, Yuehe Ding, Guangzuo Luo, et al. "Cryo-EM structure of the exocyst complex." Nature Structural & Molecular Biology 25, no. 2 (January 15, 2018): 139–46. http://dx.doi.org/10.1038/s41594-017-0016-2.

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Zhu, Xing, Dhiraj Mannar, Shanti S. Srivastava, Alison M. Berezuk, Jean-Philippe Demers, James W. Saville, Karoline Leopold, et al. "Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies." PLOS Biology 19, no. 4 (April 29, 2021): e3001237. http://dx.doi.org/10.1371/journal.pbio.3001237.

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The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

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Zeng, Lingxiao, Wei Ding, and Quan Hao. "Using cryo-electron microscopy maps for X-ray structure determination." IUCrJ 5, no. 4 (May 11, 2018): 382–89. http://dx.doi.org/10.1107/s2052252518005857.

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X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable R work/R free. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.

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Si, Dong, and Jing He. "Modeling Beta-Traces for Beta-Barrels from Cryo-EM Density Maps." BioMed Research International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/1793213.

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Cryo-electron microscopy (cryo-EM) has produced density maps of various resolutions. Althoughα-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 ofβ-strands. The variety of shapes ofβ-sheets adds the complexity ofβ-strands detection from density maps. We propose a new approach to model traces ofβ-strands forβ-barrel density regions that are extracted from cryo-EM density maps. In the test containing eightβ-barrels extracted from experimental cryo-EM density maps at 5.5 Å–8.25 Å resolution,StrandRollerdetected about 74.26% of the amino acids in theβ-strands with an overall 2.05 Å 2-way distance between the detectedβ-traces and the observed ones, if the best of the fifteen detection cases is considered.

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Cerutti, Gabriele, Yicheng Guo, Lihong Liu, Liyuan Liu, Zhening Zhang, Yang Luo, Yiming Huang, et al. "Cryo-EM structure of the SARS-CoV-2 Omicron spike." Cell Reports 38, no. 9 (March 2022): 110428. http://dx.doi.org/10.1016/j.celrep.2022.110428.

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Xia, Shiyu, Longfei Wang, Tian‐Min Fu, and Hao Wu. "Mechanism of TRPM 2 channel gating revealed by cryo‐ EM." FEBS Journal 286, no. 17 (June 10, 2019): 3333–39. http://dx.doi.org/10.1111/febs.14939.

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Lawson, Catherine L., Andriy Kryshtafovych, Paul D. Adams, Pavel V. Afonine, Matthew L. Baker, Benjamin A. Barad, Paul Bond, et al. "Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge." Nature Methods 18, no. 2 (February 2021): 156–64. http://dx.doi.org/10.1038/s41592-020-01051-w.

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AbstractThis paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

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Wang, Ivy, Sandeep K. Gupta, Guillaume Ems, Nadishka Jayawardena, Mike Strauss, and Mihnea Bostina. "Cryo-EM Structure of a Possum Enterovirus." Viruses 14, no. 2 (February 3, 2022): 318. http://dx.doi.org/10.3390/v14020318.

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Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail possum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (formerly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses.

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Kordyukova, L. V., A. V. Moiseenko, T. A. Timofeeva, and I. T. Fedyakina. "Cryo-electron microscopy of enveloped viruses using upgraded transmission electron microscope: Influenza type A, B viruses and SARS-CoV-2." Vestnik Moskovskogo universiteta. Seria 16. Biologia 78, no. 3s, 2023 (December 21, 2023): 21–26. http://dx.doi.org/10.55959/10.55959/msu0137-0952-16-78-3s-4.

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Cryo-electron microscopy (cryo-EM) is indispensable for the structural studies of enveloped viruses – dangerous pathogens of humans and animals. Yet, it requires highly specialized equipment as well as careful sample preparation. In this work, the capabilities of transmission electron microscope JEOL JEM-2100 equipped with cryo-transfer holder are used, and preliminary cryo-EM data for influenza A and B virus strains and SARS-CoV-2 inactivated with beta-propiolactone are presented. Image analysis allows: (1) to distinguish “empty” viral particles from “full” ones (containing nucleocapsid); (2) to visualize the lipid bilayer of the viral envelope; (3) identify influenza virus surface antigens and the M1 protein layer combined with the inner lipid monolayer; (4) distinguish different morphology of S-spikes on the surface of inactivated SARS-CoV-2 virions. The developed approach provides good image quality for both fundamental and applied research.

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Li, Zhuang, Heng Zhang, Renjian Xiao, Ruijie Han, and Leifu Chang. "Cryo-EM structure of the RNA-guided ribonuclease Cas12g." Nature Chemical Biology 17, no. 4 (January 25, 2021): 387–93. http://dx.doi.org/10.1038/s41589-020-00721-2.

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Singh, Arunima. "GemSpot allows modeling of ligands in cryo-EM maps." Nature Methods 17, no. 7 (July 2020): 656. http://dx.doi.org/10.1038/s41592-020-0900-2.

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Vassal-Stermann, Emilie, Stephanie Hutin, Pascal Fender, and Wim P. Burmeister. "Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein 2." Acta Crystallographica Section F Structural Biology Communications 75, no. 12 (November 27, 2019): 750–57. http://dx.doi.org/10.1107/s2053230x19015784.

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The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Å resolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction.

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Liu, Chuang, Luiza Mendonça, Yang Yang, Yuanzhu Gao, Chenguang Shen, Jiwei Liu, Tao Ni, et al. "The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET." Structure 28, no. 11 (November 2020): 1218–24. http://dx.doi.org/10.1016/j.str.2020.10.001.

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Peck, Jared V., Jonathan F. Fay, and Joshua D. Strauss. "High-speed high-resolution data collection on a 200 keV cryo-TEM." IUCrJ 9, no. 2 (January 29, 2022): 243–52. http://dx.doi.org/10.1107/s2052252522000069.

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Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (∼17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8–1.9 Å resolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 Å was obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.

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Wang, Jimin, S. Kundhavai Natchiar, Peter B. Moore, and Bruno P. Klaholz. "Identification of Mg2+ ions next to nucleotides in cryo-EM maps using electrostatic potential maps." Acta Crystallographica Section D Structural Biology 77, no. 4 (March 30, 2021): 534–39. http://dx.doi.org/10.1107/s2059798321001893.

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Cryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg2+ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg2+ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2′-O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions.

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Yamamori, Yu, and Kentaro Tomii. "Application of Homology Modeling by Enhanced Profile–Profile Alignment and Flexible-Fitting Simulation to Cryo-EM Based Structure Determination." International Journal of Molecular Sciences 23, no. 4 (February 10, 2022): 1977. http://dx.doi.org/10.3390/ijms23041977.

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Application of cryo-electron microscopy (cryo-EM) is crucially important for ascertaining the atomic structure of large biomolecules such as ribosomes and protein complexes in membranes. Advances in cryo-EM technology and software have made it possible to obtain data with near-atomic resolution, but the method is still often capable of producing only a density map with up to medium resolution, either partially or entirely. Therefore, bridging the gap separating the density map and the atomic model is necessary. Herein, we propose a methodology for constructing atomic structure models based on cryo-EM maps with low-to-medium resolution. The method is a combination of sensitive and accurate homology modeling using our profile–profile alignment method with a flexible-fitting method using molecular dynamics simulation. As described herein, this study used benchmark applications to evaluate the model constructions of human two-pore channel 2 (one target protein in CASP13 with its structure determined using cryo-EM data) and the overall structure of Enterococcus hirae V-ATPase complex.

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Casasanta, Michael, G. M. Jonaid, Liam Kaylor, William Luqiu, Maria Solares, Mariah Schroen, William Dearnaley, Jarad Wilson, Madelin Dukes, and Deborah Kelly. "Cryo-EM structural analysis of the SARS-CoV-2 Nucleocapsid protein." Microscopy and Microanalysis 27, S1 (July 30, 2021): 1378–80. http://dx.doi.org/10.1017/s1431927621005134.

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Kern, David M., Ben Sorum, Sonali S. Mali, Christopher M. Hoel, Savitha Sridharan, Jonathan P. Remis, Daniel B. Toso, Abhay Kotecha, Diana M. Bautista, and Stephen G. Brohawn. "Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs." Nature Structural & Molecular Biology 28, no. 7 (June 22, 2021): 573–82. http://dx.doi.org/10.1038/s41594-021-00619-0.

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Wang, Jia, Xianqiang Song, Dandan Zhang, Xiaoqing Chen, Xun Li, Yaping Sun, Cui Li, et al. "Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism." Cell Research 30, no. 5 (February 11, 2020): 436–45. http://dx.doi.org/10.1038/s41422-020-0280-2.

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Scheres, Sjors H. W., Benjamin Ryskeldi-Falcon, and Michel Goedert. "Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids." Nature 621, no. 7980 (September 27, 2023): 701–10. http://dx.doi.org/10.1038/s41586-023-06437-2.

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Xu, Cong, Wenyu Han, and Yao Cong. "Cryo-EM and cryo-ET of the spike, virion, and antibody neutralization of SARS-CoV-2 and VOCs." Current Opinion in Structural Biology 82 (October 2023): 102664. http://dx.doi.org/10.1016/j.sbi.2023.102664.

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Pintilie, Grigore, and Wah Chiu. "Validation, analysis and annotation of cryo-EM structures." Acta Crystallographica Section D Structural Biology 77, no. 9 (August 31, 2021): 1142–52. http://dx.doi.org/10.1107/s2059798321006069.

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The process of turning 2D micrographs into 3D atomic models of the imaged macromolecules has been under rapid development and scrutiny in the field of cryo-EM. Here, some important methods for validation at several stages in this process are described. Firstly, how Fourier shell correlation of two independent maps and phase randomization beyond a certain frequency address the assessment of map resolution is reviewed. Techniques for local resolution estimation and map sharpening are also touched upon. The topic of validating models which are either built de novo or based on a known atomic structure fitted into a cryo-EM map is then approached. Map–model comparison using Q-scores and Fourier shell correlation plots is used to assure the agreement of the model with the observed map density. The importance of annotating the model with B factors to account for the resolvability of individual atoms in the map is illustrated. Finally, the timely topic of detecting and validating water molecules and metal ions in maps that have surpassed ∼2 Å resolution is described.

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Wieferig, Jan-Philip, Deryck J. Mills, and Werner Kühlbrandt. "Devitrification reduces beam-induced movement in cryo-EM." IUCrJ 8, no. 2 (March 1, 2021): 186–94. http://dx.doi.org/10.1107/s2052252520016243.

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As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å−2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.

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Ognjenović, Jana, Reinhard Grisshammer, and Sriram Subramaniam. "Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies." Annual Review of Biomedical Engineering 21, no. 1 (June 4, 2019): 395–415. http://dx.doi.org/10.1146/annurev-bioeng-060418-052453.

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In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

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Wang, Lei, Jun Xu, Sheng Cao, Dapeng Sun, Heng Liu, Qiuyuan Lu, Zheng Liu, Yang Du, and Cheng Zhang. "Cryo-EM structure of the AVP–vasopressin receptor 2–Gs signaling complex." Cell Research 31, no. 8 (March 4, 2021): 932–34. http://dx.doi.org/10.1038/s41422-021-00483-z.

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Kimanius, Dari, Björn Forsberg, and Erik Lindahl. "Accelerated Cryo-EM Structure Determination with Parallelisation using GPUs in Relion-2." Biophysical Journal 112, no. 3 (February 2017): 575a. http://dx.doi.org/10.1016/j.bpj.2016.11.3096.

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Ke, Zunlong, Joaquin Oton, Kun Qu, Sjors H. W. Scheres, and John A. G. Briggs. "Structures, Distributions, and Conformations of SARS-CoV-2 Spike Proteins on Intact Virions by Cryo-EM and Cryo-ET." Microscopy and Microanalysis 29, Supplement_1 (July 22, 2023): 902–3. http://dx.doi.org/10.1093/micmic/ozad067.448.

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Yuan, Shuai, Jialing Wang, Dongjie Zhu, Nan Wang, Qiang Gao, Wenyuan Chen, Hao Tang, et al. "Cryo-EM structure of a herpesvirus capsid at 3.1 Å." Science 360, no. 6384 (April 5, 2018): eaao7283. http://dx.doi.org/10.1126/science.aao7283.

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Structurally and genetically, human herpesviruses are among the largest and most complex of viruses. Using cryo–electron microscopy (cryo-EM) with an optimized image reconstruction strategy, we report the herpes simplex virus type 2 (HSV-2) capsid structure at 3.1 angstroms, which is built up of about 3000 proteins organized into three types of hexons (central, peripentonal, and edge), pentons, and triplexes. Both hexons and pentons contain the major capsid protein, VP5; hexons also contain a small capsid protein, VP26; and triplexes comprise VP23 and VP19C. Acting as core organizers, VP5 proteins form extensive intermolecular networks, involving multiple disulfide bonds (about 1500 in total) and noncovalent interactions, with VP26 proteins and triplexes that underpin capsid stability and assembly. Conformational adaptations of these proteins induced by their microenvironments lead to 46 different conformers that assemble into a massive quasisymmetric shell, exemplifying the structural and functional complexity of HSV.

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Nešić, Dragana, Martin Bush, Aleksandar Spasic, Jihong Li, Tetsuji Kamata, Makoto Handa, Marta Filizola, Thomas Walz, and Barry S. Coller. "Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding." Blood Advances 5, no. 7 (March 24, 2021): 1781–90. http://dx.doi.org/10.1182/bloodadvances.2020004166.

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The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp–binding pocket. To elucidate the mechanism, we studied the αIIbβ3–PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces αIIbβ3 ligand binding by binding to extended conformations and by preventing the interactions between the αIIb and β3 leg domains and subsequently the βI and β3 leg domains required for the bent-closed conformation.

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Fibriansah, Guntur, Kristie D. Ibarra, Thiam-Seng Ng, Scott A. Smith, Joanne L. Tan, Xin-Ni Lim, Justin S. G. Ooi, et al. "Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers." Science 349, no. 6243 (July 2, 2015): 88–91. http://dx.doi.org/10.1126/science.aaa8651.

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There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

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Kukimoto-Niino, Mutsuko, Kazushige Katsura, Rahul Kaushik, Haruhiko Ehara, Takeshi Yokoyama, Tomomi Uchikubo-Kamo, Reiko Nakagawa, et al. "Cryo-EM structure of the human ELMO1-DOCK5-Rac1 complex." Science Advances 7, no. 30 (July 2021): eabg3147. http://dx.doi.org/10.1126/sciadv.abg3147.

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The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) promotes cell motility, phagocytosis, and cancer metastasis through activation of Rho guanosine triphosphatases. Engulfment and cell motility (ELMO) proteins are binding partners of DOCK and regulate Rac activation. Here, we report the cryo–electron microscopy structure of the active ELMO1-DOCK5 complex bound to Rac1 at 3.8-Å resolution. The C-terminal region of ELMO1, including the pleckstrin homology (PH) domain, aids in the binding of the catalytic DOCK homology region 2 (DHR-2) domain of DOCK5 to Rac1 in its nucleotide-free state. A complex α-helical scaffold between ELMO1 and DOCK5 stabilizes the binding of Rac1. Mutagenesis studies revealed that the PH domain of ELMO1 enhances the GEF activity of DOCK5 through specific interactions with Rac1. The structure provides insights into how ELMO modulates the biochemical activity of DOCK and how Rac selectivity is achieved by ELMO.

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Mannar, Dhiraj, James W. Saville, Xing Zhu, Shanti S. Srivastava, Alison M. Berezuk, Katharine S. Tuttle, Ana Citlali Marquez, Inna Sekirov, and Sriram Subramaniam. "SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein–ACE2 complex." Science 375, no. 6582 (February 18, 2022): 760–64. http://dx.doi.org/10.1126/science.abn7760.

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The newly reported Omicron variant is poised to replace Delta as the most prevalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant across the world. Cryo–electron microscopy (cryo-EM) structural analysis of the Omicron variant spike protein in complex with human angiotensin-converting enzyme 2 (ACE2) reveals new salt bridges and hydrogen bonds formed by mutated residues arginine-493, serine-496, and arginine-498 in the receptor binding domain with ACE2. These interactions appear to compensate for other Omicron mutations such as the substitution of asparagine for lysine at position 417 (K417N) that are known to reduce ACE2 binding affinity, resulting in similar biochemical ACE2 binding affinities for the Delta and Omicron variants. Neutralization assays show that pseudoviruses that display the Omicron spike protein exhibit increased antibody evasion. The increase in antibody evasion and the retention of strong interactions at the ACE2 interface thus represent important molecular features that likely contribute to the rapid spread of the Omicron variant.

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Na, C. L., H. K. Hagler, and K. H. Muntz. "Development of a cryosection EM autoradiography technique and its application for the subcellular localization of receptors." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 920–21. http://dx.doi.org/10.1017/s0424820100167068.

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Recent progress in immunocytochemistry and cryo-techniques has made it possible to study receptor localization at the subcellular level. For many receptor-ligand systems suitable antibodies are not available and it would be more appropriate to use radioligands to study these receptors. Although fresh frozen sections have been widely used in light microscopy (LM) autoradiography studies, to our knowledge, no one has established a technique using electron microscope (EM) autoradiography with ultrathin frozen sections.Unlike conventional EM approaches which can extract many biological molecules during dehydration and plastic embedding steps, we have adopted the method of Tokuyasu combined with LM autoradiography protocol for frozen sections to develop a new EM autoradiography technique using ultrathin frozen sections. Heart blocks were fixed in 2% periodate-lysine-paraformaldehyde (PLP) and 0.1% glutaraldehyde (GA), sucrose infused, and frozen in liquid nitrogen. They were sectioned in a Reichert Ultracut S cryo-microtome equipped with a Reichert FCS cryo-unit.

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Valimehr, Sepideh, Rémi Vuillemot, Mohsen Kazemi, Slavica Jonic, and Isabelle Rouiller. "Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes." International Journal of Molecular Sciences 25, no. 6 (March 16, 2024): 3371. http://dx.doi.org/10.3390/ijms25063371.

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Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.

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Kostenko, Anastasiya, Konstantin Palamarchuk, Yury Chesnokov, Konstantin Plokhikh, Tatyana Bukreeva, and Roman Kamyshinsky. "Abstract P-34: Cryo-EM Study of Submicrocapsules with a Shell of Nanoparticle Heteroaggregates and Polyelectrolyte Layers." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S26—S27. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p34.

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Background: Currently, different approaches of active and passive targeted drug delivery are being developed. One of the most promising methods of targeted drug delivery is the use of capsules. For instance, colloidosomes—capsules consisting of the shell formed by colloidal particles at the interface of the emulsion—can be used for targeted delivery of antitumor drugs or any other drugs in liquid form. Here we present results of cryo-EM study of submicrocapsules with the soybean oil core and with the shell consisting of SiO2 nanoparticles and detonation nanodiamonds (DNDs) stabilized with chitosan and alginate. Methods: Сryo-electron tomography (Cryo-ET) was used to identify the morphological features of the submicrocapsules. Preliminary screening of samples and cryo-ET data collection were performed using Titan Krios cryo-EM (ThermoFisher Scientific, US) equipped with Falcon 2 direct electron detector. The restoration of the tomographic series was carried out using IMOD software. Eman2 was used for segmentation and UCSF Chimera was used for visualization of the 3D model. Submicron capsules were obtained by stabilizing oil droplets with a mixture of SiO2 nanoparticles and DNDs. To form a stable shell, an additional layer of silica particles and polyelectrolyte layers of alginate/chitosan were applied to the droplets of the dispersed phase of the emulsion by physical adsorption. Results: Cryo-EM data showed the presence of submicrocapsules with a diameter in the range of 200-900 nm. Although a significant fraction of submicrocapsules was found to be partially destroyed, results of cryo-ET study of intact capsules demonstrated that silicon dioxide nanoparticles form a net, while DNDs form clusters. Conclusion: Here we demonstrate the results of the study of submicron capsules with a shell of silica nanoparticles and DNDs. It was found that a uniform distribution of DNDs is not a prerequisite for the creation of submicron capsules that contradicts the theoretical model.

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Malhotra, Sony, Martyn Winn, and Agnel Praveen Joseph. "Validation of cryo-EM structures of SARS-CoV-2 and mapping genomic mutations." Acta Crystallographica Section A Foundations and Advances 77, a2 (August 14, 2021): C615. http://dx.doi.org/10.1107/s0108767321090796.

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Bertram, Karl, Dmitry E. Agafonov, Wen-Ti Liu, Olexandr Dybkov, Cindy L. Will, Klaus Hartmuth, Henning Urlaub, Berthold Kastner, Holger Stark, and Reinhard Lührmann. "Cryo-EM structure of a human spliceosome activated for step 2 of splicing." Nature 542, no. 7641 (January 11, 2017): 318–23. http://dx.doi.org/10.1038/nature21079.

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Yu, Zhiheng, Malgorzata D. Gonciarz, Wesley I. Sundquist, Christopher P. Hill, and Grant J. Jensen. "Cryo-EM Structure of Dodecameric Vps4p and Its 2:1 Complex with Vta1p." Journal of Molecular Biology 377, no. 2 (March 2008): 364–77. http://dx.doi.org/10.1016/j.jmb.2008.01.009.

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Kong, Leopold, Kem A. Sochacki, Huaibin Wang, Shunming Fang, Bertram Canagarajah, Andrew D. Kehr, William J. Rice, Marie-Paule Strub, Justin W. Taraska, and Jenny E. Hinshaw. "Author Correction: Cryo-EM of the dynamin polymer assembled on lipid membrane." Nature 564, no. 7734 (October 30, 2018): E6. http://dx.doi.org/10.1038/s41586-018-0612-2.

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Mears, J., F. Alvarez, L. Zhou, S. Fang, and J. Hinshaw. "Cryo-EM studies of dynamin-related proteins that regulate mitochondrial fission." Microscopy and Microanalysis 18, S2 (July 2012): 60–61. http://dx.doi.org/10.1017/s1431927612002152.

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Caran, K. L., R. P. Apkarian, and F. M. Menger. "Examination of Large Unilamellar Vesicles (Luvs) Using Cryo-hrsem and Cryo-Stem." Microscopy and Microanalysis 5, S2 (August 1999): 1210–11. http://dx.doi.org/10.1017/s1431927600019371.

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Vesicles derive their structure from the aqueous environment surrounding and within them. It follows that traditional EM specimen preparation techniques of fixation, dehydration and drying can induce a variety of morphological changes to the membranes. Vitrification of aqueous suspensions of vesicles provides a means for observation of these structures in their fully hydrated unfixed state. Common cryogenic techniques for the visualization of these and other colloidal particles include cryo-TEM of vitrified thin films and freeze-fracture TEM (FF-TEM) of platinum replicas. We report the use of cryo-high resolution SEM (cryo- HRSEM) and cryo-STEM for the study of the morphology of membrane features of synthetic extruded vesicles.LUVs were prepared by the extrusion method. Dried lipid films of l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) were hydrated with 0.5 mL of Milli-Q water and stirred for 10 minutes to make suspensions (10 mM in lipids) which were extruded 19 times through a polycarbonate filter with 100 nm pores.

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Bock, Lars V., and Helmut Grubmüller. "Effects of cryo-EM cooling on structural ensembles." Nature Communications 13, no. 1 (March 31, 2022). http://dx.doi.org/10.1038/s41467-022-29332-2.

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AbstractStructure determination by cryo electron microscopy (cryo-EM) provides information on structural heterogeneity and ensembles at atomic resolution. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to cryogenic temperatures. To what extent the structural ensemble is perturbed during cooling is currently unknown. Here, to quantify the effects of cooling, we combined continuum model calculations of the temperature drop, molecular dynamics simulations of a ribosome complex before and during cooling with kinetic models. Our results suggest that three effects markedly contribute to the narrowing of the structural ensembles: thermal contraction, reduced thermal motion within local potential wells, and the equilibration into lower free-energy conformations by overcoming separating free-energy barriers. During cooling, barrier heights below 10 kJ/mol were found to be overcome, which is expected to reduce B-factors in ensembles imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures.

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Dhakal, Ashwin, Rajan Gyawali, Liguo Wang, and Jianlin Cheng. "A large expert-curated cryo-EM image dataset for machine learning protein particle picking." Scientific Data 10, no. 1 (June 22, 2023). http://dx.doi.org/10.1038/s41597-023-02280-2.

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AbstractCryo-electron microscopy (cryo-EM) is a powerful technique for determining the structures of biological macromolecular complexes. Picking single-protein particles from cryo-EM micrographs is a crucial step in reconstructing protein structures. However, the widely used template-based particle picking process is labor-intensive and time-consuming. Though machine learning and artificial intelligence (AI) based particle picking can potentially automate the process, its development is hindered by lack of large, high-quality labelled training data. To address this bottleneck, we present CryoPPP, a large, diverse, expert-curated cryo-EM image dataset for protein particle picking and analysis. It consists of labelled cryo-EM micrographs (images) of 34 representative protein datasets selected from the Electron Microscopy Public Image Archive (EMPIAR). The dataset is 2.6 terabytes and includes 9,893 high-resolution micrographs with labelled protein particle coordinates. The labelling process was rigorously validated through 2D particle class validation and 3D density map validation with the gold standard. The dataset is expected to greatly facilitate the development of both AI and classical methods for automated cryo-EM protein particle picking.

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Zhang, Hang, Shiyu Wang, Zhenzhen Zhang, Mengzhuo Hou, Chunyu Du, Zhenye Zhao, Horst Vogel, et al. "Cryo-EM structure of human heptameric pannexin 2 channel." Nature Communications 14, no. 1 (March 3, 2023). http://dx.doi.org/10.1038/s41467-023-36861-x.

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AbstractPannexin 2 (Panx2) is a large-pore ATP-permeable channel with critical roles in various physiological processes, such as the inflammatory response, energy production and apoptosis. Its dysfunction is related to numerous pathological conditions including ischemic brain injury, glioma and glioblastoma multiforme. However, the working mechanism of Panx2 remains unclear. Here, we present the cryo-electron microscopy structure of human Panx2 at a resolution of 3.4 Å. Panx2 structure assembles as a heptamer, forming an exceptionally wide channel pore across the transmembrane and intracellular domains, which is compatible with ATP permeation. Comparing Panx2 with Panx1 structures in different states reveals that the Panx2 structure corresponds to an open channel state. A ring of seven arginine residues located at the extracellular entrance forms the narrowest site of the channel, which serves as the critical molecular filter controlling the permeation of substrate molecules. This is further verified by molecular dynamics simulations and ATP release assays. Our studies reveal the architecture of the Panx2 channel and provide insights into the molecular mechanism of its channel gating.

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Bodakuntla, Satish, Christopher Cyrus Kuhn, Christian Biertümpfel, and Naoko Mizuno. "Cryo-electron microscopy in the fight against COVID-19—mechanism of virus entry." Frontiers in Molecular Biosciences 10 (October 6, 2023). http://dx.doi.org/10.3389/fmolb.2023.1252529.

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Cryogenic electron microscopy (cryo-EM) and electron tomography (cryo-ET) have become a critical tool for studying viral particles. Cryo-EM has enhanced our understanding of viral assembly and replication processes at a molecular resolution. Meanwhile, in situ cryo-ET has been used to investigate how viruses attach to and invade host cells. These advances have significantly contributed to our knowledge of viral biology. Particularly, prompt elucidations of structures of the SARS-CoV-2 spike protein and its variants have directly impacted the development of vaccines and therapeutic measures. This review discusses the progress made by cryo-EM based technologies in comprehending the severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2), the virus responsible for the devastating global COVID-19 pandemic in 2020 with focus on the SARS-CoV-2 spike protein and the mechanisms of the virus entry and replication.

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Journal articles on the topic 'Cryo EM 2'

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