High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity

Tao Ni, Thomas Frosio, Luiza Mendonça, Yuewen Sheng, Daniel Clare, Benjamin A. Himes, Peijun Zhang

Published: 2022-01-11 DOI: 10.1038/s41596-021-00648-5

3D density maps

cryo-electron tomography

subtomogram averaging

emClarity

macromolecular complexes

AI 解读

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Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes insitu and in cellular context at or below subnanometer resolution and has led to unprecedented insights in
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ACKNOWLEDGMENTS

Zhang, P. Advances in cryo-electron tomography and subtomogram averaging and classification. Curr. Opin. Struct. Biol. 58, 249–258 (2019).
Kaplan, M. et al. In situ imaging and structure determination of biomolecular complexes using electron cryo-tomography. Methods Mol. Biol. 2215, 83–111 (2021).
Turk, M. & Baumeister, W. The promise and the challenges of cryo-electron tomography. FEBS Lett. 594, 3243–3261 (2020).
Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016).
Forster, F. & Hegerl, R. Structure determination in situ by averaging of tomograms. Methods Cell Biol. 79, 741–767 (2007).
Bykov, Y. S. et al. The structure of the COPI coat determined within the cell. eLife http://doi.org/10.7554/eLife.32493 (2017).
Zhang, Y. et al. Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex. Cell Res. 30, 532–540 (2020).
Pfeffer, S. et al. Structure of the native Sec61 protein-conducting channel. Nat. Commun. 6, 8403 (2015).
Cassidy, C. K. et al. CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling. eLife http://doi.org/10.7554/eLife.08419 (2015).
Dodonova, S. O., Prinz, S., Bilanchone, V., Sandmeyer, S. & Briggs, J. A. G. Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses. Proc. Natl Acad. Sci. USA 116, 10048–10057 (2019).
Mattei, S., Glass, B., Hagen, W. J., Krausslich, H. G. & Briggs, J. A. The structure and flexibility of conical HIV-1 capsids determined within intact virions. Science 354, 1434–1437 (2016).
Dick, R. A. et al. Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly. PLoS Pathog. 16, e1008277 (2020).
Schur, F. K. et al. An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation. Science 353, 506–508 (2016).
Qu, K. et al. Structure and architecture of immature and mature murine leukemia virus capsids. Proc. Natl Acad. Sci. USA 115, E11751–E11760 (2018).
von Kugelgen, A. et al. In situ structure of an intact lipopolysaccharide-bound bacterial surface layer. Cell 180, 348–358 e315 (2020).
Tegunov, D., Xue, L., Dienemann, C., Cramer, P. & Mahamid, J. Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 A in cells. Nat. Methods 18, 186–193 (2021).
Lucic, V., Rigort, A. & Baumeister, W. Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419 (2013).
Wan, W. & Briggs, J. A. Cryo-electron tomography and subtomogram averaging. Methods Enzymol. 579, 329–367 (2016).
Turonova, B., Schur, F. K. M., Wan, W. & Briggs, J. A. G. Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4A. J. Struct. Biol. 199, 187–195 (2017).
Heumann, J. M., Hoenger, A. & Mastronarde, D. N. Clustering and variance maps for cryo-electron tomography using wedge-masked differences. J. Struct. Biol. 175, 288–299 (2011).
Nicastro, D. et al. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948 (2006).
Chen, M. et al. Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nat. Methods 14, 983–985 (2017).
Galaz-Montoya, J. G., Flanagan, J., Schmid, M. F. & Ludtke, S. J. Single particle tomography in EMAN2. J. Struct. Biol. 190, 279–290 (2015).
Galaz-Montoya, J. G. et al. Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography. J. Struct. Biol. 194, 383–394 (2016).
Bharat, T. A. & Scheres, S. H. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat. Protoc. 11, 2054–2065 (2016).
Bharat, T. A. M., Russo, C. J., Lowe, J., Passmore, L. A. & Scheres, S. H. W. Advances in single-particle electron cryomicroscopy structure determination applied to sub-tomogram averaging. Structure 23, 1743–1753 (2015).
Castano-Diez, D., Kudryashev, M., Arheit, M. & Stahlberg, H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J. Struct. Biol. 178, 139–151 (2012).
Maurer, U. E. et al. The structure of herpesvirus fusion glycoprotein B-bilayer complex reveals the protein–membrane and lateral protein–protein interaction. Structure 21, 1396–1405 (2013).
Forster, F., Pruggnaller, S., Seybert, A. & Frangakis, A. S. Classification of cryo-electron sub-tomograms using constrained correlation. J. Struct. Biol. 161, 276–286 (2008).
Hrabe, T. et al. PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis. J. Struct. Biol. 178, 177–188 (2012).
Winkler, H. 3D reconstruction and processing of volumetric data in cryo-electron tomography. J. Struct. Biol. 157, 126–137 (2007).
Himes, B. A. & Zhang, P. emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. Nat. Methods 15, 955–961 (2018).
Liu, C. et al. The architecture of inactivated SARS-CoV-2 with postfusion spikes revealed by cryo-EM and cryo-ET. Structure 28, 1218–1224 e1214 (2020).
Watanabe, R. et al. The in situ structure of Parkinson’s disease-linked LRRK2. Cell 182, 1508–1518 e1516 (2020).
Sutton, G. et al. Assembly intermediates of orthoreovirus captured in the cell. Nat. Commun. 11, 4445 (2020).
Tan, T. Y. et al. Capsid protein structure in Zika virus reveals the flavivirus assembly process. Nat. Commun. 11, 895 (2020).
Unchwaniwala, N. et al. Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication. Proc. Natl Acad. Sci. USA 117, 18680–18691 (2020).
Gibson, K. H. et al. An asymmetric sheath controls flagellar supercoiling and motility in the leptospira spirochete. eLife http://doi.org/10.7554/eLife.53672 (2020).
Cassidy, C. K. et al. Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations. Commun. Biol. 3, 24 (2020).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113 (2017).
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. eLife http://doi.org/10.7554/eLife.35383 (2018).
Heymann, J. B. Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs. Protein Sci. 27, 159–171 (2018).
Mendonca, L. et al. CryoET structures of immature HIV Gag reveal six-helix bundle. Commun. Biol. 4, 481 (2021).

High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity

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