Cryo-EM in drug discovery: Achievements, limitations and prospects

Nature Reviews Drug Discovery

Volumn 17, Issue 7, 2018, Pages 471-492

  Renaud, Jean Paul ^a,b,c   Chari, Ashwin ^d   Ciferri, Claudio ^e   Liu, Wen Ti ^a   Rémigy, Hervé William ^f   Stark, Holger ^d   Wiesmann, Christian ^g  

^a Building A: Chemistry   (France)

^b RiboStruct   (France)

^c UNIVERSITÉ DE STRASBOURG   (France)

^d MAX PLANCK INSTITUTE FOR BIOPHYSICAL CHEMISTRY   (Germany)

^e GENENTECH INC   (United States)

^f Thermo Fisher Scientific   (United States)

^g NOVARTIS PHARMA AG   (Switzerland)

Author keywords

[No Author keywords available]

Indexed keywords

CALNEXIN; G PROTEIN COUPLED RECEPTOR; GAMMA SECRETASE INHIBITOR; GLUCAGON LIKE PEPTIDE 1 RECEPTOR; GLYCOPROTEIN; MEMBRANE PROTEIN; MONOCLONAL ANTIBODY; VANILLOID RECEPTOR 1;

CRYOELECTRON MICROSCOPY; CRYSTALLOGRAPHY; HUMAN; IMAGE ANALYSIS; IMAGE PROCESSING; MACROMOLECULE; MOLECULAR WEIGHT; NONHUMAN; PRIORITY JOURNAL; PROCESS OPTIMIZATION; REVIEW; ANIMAL; CHEMISTRY; DEVICES; DRUG DEVELOPMENT; PROCEDURES; X RAY CRYSTALLOGRAPHY;

ANIMALS; CRYOELECTRON MICROSCOPY; CRYSTALLOGRAPHY, X-RAY; DRUG DISCOVERY; MACROMOLECULAR SUBSTANCES;

EID: 85049235848     PISSN: 14741776     EISSN: 14741784     Source Type: Journal    
DOI: 10.1038/nrd.2018.77     Document Type: Review

References (302)

1. von Borries, B. E. &Ruska, H. R. Bakterien und Virus in übermikroskopischer Aufnahme. J. Mol. Med. 17, 921-925 (1938).
2. Kausche, G. A., Pfankuch, E. &Ruska, H. Die Sichtbarmachung von pflanzlichem Virus im Übermikroskop. Naturwissenschaften 27, 292-299 (1939).
3. Kruger, D. H., Schneck, P. &Gelderblom, H. R. Helmut Ruska and the visualisation of viruses. Lancet 355, 1713-1717 (2000).
4. Dubochet, J. &McDowall, A. W. Vitrification of pure water for electron microscopy. J. Microsc. 124, RP3-RP4 (1981).
5. Dubochet, J., Lepault, J., Freeman, R., Berriman, J. A. &Homo, J.-C. Electron microscopy of frozen water and aqueous solutions. J. Microsc. 128, 219-237 (1982).
6. Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J. &McDowall, A. W. Frozen aqueous suspensions. Ultramicroscopy 10, 55-62 (1982).
7. Adrian, M., Dubochet, J., Lepault, J. &McDowall, A. W. Cryo-electron microscopy of viruses. Nature 308, 32-36 (1984).
8. van Heel, M. &Frank, J. Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6, 187-194 (1981).
9. Frank, J., Shimkin, B. &Dowse, H. Spider-A modular software system for electron image processing. Ultramicroscopy 6, 343-357 (1981).
10. van Heel, M. &Keegstra, W. IMAGIC: a fast, flexible and friendly image analysis software system. Ultramicroscopy 7, 113-129 (1981).
11. Penczek, P., Radermacher, M. &Frank, J. Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40, 33-53 (1992).
12. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190-199 (1996).
13. Marabini, R. et al. Xmipp: an image processing package for electron microscopy. J. Struct. Biol. 116, 237-240 (1996).
14. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41-60 (2005).
15. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36-51 (2005).
16. Lander, G. C. et al. Appion: an integrated, databasedriven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95-102 (2009).
17. van Heel, M. et al. in International Tables for Crystallography, Volume F, 2nd Edition, Crystallography of Biological Macromolecules (eds Arnold, E., Himmel, D. M. &Rossmann, M. G.) 624-628 (Wiley, 2012).
18. Kimanius, D., Forsberg, B. O., Scheres, S. H. &Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).
19. Bell, J. M., Chen, M., Baldwin, P. R. &Ludtke, S. J. High resolution single particle refinement in EMAN2.1. Methods 100, 25-34 (2016).
20. Punjani, A., Rubinstein, J. L., Fleet, D. J. &Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290-296 (2017).
21. Grant, T. Rohou, A. &Grigorieff, N. cisTEM, userfriendly software for single-particle image processing. eLife 7, e35383 (2018).
22. Kuhlbrandt, W. Biochemistry. The resolution revolution. Science 343, 1443-1444 (2014).
23. Bai, X. C., McMullan, G. &Scheres, S. H. How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40, 49-57 (2015).
24. Khoshouei, M., Radjainia, M., Baumeister, W. &Danev, R. Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate. Nat. Commun. 8, 16099 (2017).
25. Merk, A. et al. Breaking cryo-EM resolution barriers to facilitate drug discovery. Cell 165, 1698-1707 (2016).
26. Blundell, T. L. &Patel, S. High-throughput X-ray crystallography for drug discovery. Curr. Opin. Pharmacol. 4, 490-496 (2004).
27. Blundell, T. L. et al. Structural biology and bioinformatics in drug design: opportunities and challenges for target identification and lead discovery. Phil. Trans. R. Soc. B 361, 413-423 (2006).
28. Pellecchia, M. et al. Perspectives on NMR in drug discovery: a technique comes of age. Nat. Rev. Drug Discov. 7, 738-745 (2008).
29. Zheng, H. et al. X-Ray crystallography over the past decade for novel drug discovery-where are we heading next? Expert Opin. Drug Discov. 10, 975-989 (2015).
30. Campbell, M. G. et al. Movies of ice-embedded particles enhance resolution in electron cryomicroscopy. Structure 20, 1823-1828 (2012).
31. Grant, T. &Grigorieff, N. Automatic estimation and correction of anisotropic magnification distortion in electron microscopes. J. Struct. Biol. 192, 204-208 (2015).
32. Brilot, A. F. et al. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177, 630-637 (2012).
33. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331-332 (2017).
34. Russo, C. J. &Passmore, L. A. Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377-1380 (2014).
35. Russo, C. J. &Passmore, L. A. Robust evaluation of 3D electron cryomicroscopy data using tilt-pairs. J. Struct. Biol. 187, 112-118 (2014).
36. Stark, H., Zemlin, F. &Boettcher, C. Electron radiation damage to protein crystals of bacteriorhodopsin at different temperatures. Ultramicroscopy 63, 75-79 (1996).
37. Grant, T. &Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 A reconstruction of rotavirus VP6. eLife 4, e06980 (2015).
38. Radermacher, M., Wagenknecht, T., Verschoor, A. &Frank, J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc. 146, 113-136 (1987).
39. Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117-125 (2007).
40. Reboul, C. F., Eager, M., Elmlund, D. &Elmlund, H. Single-particle cryo-EM-Improved ab initio 3D reconstruction with SIMPLE/PRIME. Protein Sci. 27, 51-61 (2018).
41. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530 (2012).
42. Unverdorben, P. et al. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome. Proc. Natl Acad. Sci. USA 111, 5544-5549 (2014).
43. Bai, X. C., Rajendra, E., Yang, G., Shi, Y. &Scheres, S. H. Sampling the conformational space of the catalytic subunit of human gamma-secretase. eLife 4, e11182 (2015).
44. von Loeffelholz, O. et al. Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes. Curr. Opin. Struct. Biol. 46, 140-148 (2017).
45. Tagare, H. D., Barthel, A. &Sigworth, F. J. An adaptive expectation-maximization algorithm with GPU implementation for electron cryomicroscopy. J. Struct. Biol. 171, 256-265 (2010).
46. Glaeser, R. M. &Hall, R. J. Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects. Biophys. J. 100, 2331-2337 (2011).
47. Thompson, R. F., Walker, M., Siebert, C. A., Muench, S. P. &Ranson, N. A. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology. Methods 100, 3-15 (2016).
48. Zernike, F. How I discovered phase contrast. Science 121, 345-349 (1955).
49. Danev, R., Buijsse, B., Khoshouei, M., Plitzko, J. M. &Baumeister, W. Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc. Natl Acad. Sci. USA 111, 15635-15640 (2014).
50. Danev, R., Tegunov, D. &Baumeister, W. Using the Volta phase plate with defocus for cryo-EM single particle analysis. eLife 6, e23006 (2017).
51. Danev, R. &Baumeister, W. Expanding the boundaries of cryo-EM with phase plates. Curr. Opin. Struct. Biol. 46, 87-94 (2017).
52. Wu, S. et al. Fabs enable single particle cryoEM studies of small proteins. Structure 20, 582-592 (2012).
53. Liu, Y., Gonen, S., Gonen, T. &Yeates, T. O. Nearatomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. Proc. Natl Acad. Sci. USA 115, 3362-3367 (2018).
54. Chari, A. et al. ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space. Nat. Methods 12, 859-865 (2015).
55. Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53-55 (2008).
56. Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N. &Nordlund, P. Thermofluor-based highthroughput stability optimization of proteins for structural studies. Anal Biochem. 357, 289-298 (2006).
57. Boivin, S., Kozak, S. &Meijers, R. Optimization of protein purification and characterization using thermofluor screens. Protein Expr. Purif. 91, 192-206 (2013).
58. Benjamin, C. J. et al. Nonfouling NTA-PEG-based TEM grid coatings for selective capture of histidinetagged protein targets from cell lysates. Langmuir 32, 551-559 (2016).
59. Benjamin, C. J. et al. Selective capture of histidinetagged proteins from cell lysates using TEM grids modified with NTA-graphene oxide. Sci. Rep. 6, 32500 (2016).
60. Yu, G., Li, K. &Jiang, W. Antibody-based affinity cryo-EM grid. Methods 100, 16-24 (2016).
61. Yu, G., Li, K., Huang, P., Jiang, X. &Jiang, W. Antibody-based affinity cryoelectron microscopy at 2.6-A resolution. Structure 24, 1984-1990 (2016).
62. Russo, C. J. &Passmore, L. A. Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas. Nat. Methods 11, 649-652 (2014).
63. Sader, K., Stopps, M., Calder, L. J. &Rosenthal, P. B. Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electronoptical effects of charging. J. Struct. Biol. 183, 531-536 (2013).
64. Pantelic, R. S., Meyer, J. C., Kaiser, U., Baumeister, W. &Plitzko, J. M. Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples. J. Struct. Biol. 170, 152-156 (2010).
65. Snijder, J. et al. Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids. J. Struct. Biol. 198, 38-42 (2017).
66. Adrian, M., Dubochet, J., Fuller, S. D. &Harris, J. R. Cryo-negative staining. Micron 29, 145-160 (1998).
67. Heymann, J. B., Cardone, G., Winkler, D. C. &Steven, A. C. Computational resources for cryoelectron tomography in Bsoft. J. Struct. Biol. 161, 232-242 (2008).
68. Korinek, A., Beck, F., Baumeister, W., Nickell, S. &Plitzko, J. M. Computer controlled cryo-electron microscopy-TOM(2) a software package for highthroughput applications. J. Struct. Biol. 175, 394-405 (2011).
69. Coudray, N. et al. Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis. J. Struct. Biol. 173, 365-374 (2011).
70. Harris, J. R. &Adrian, M. Preparation of thin-film frozen-hydrated/vitrified biological specimens for cryoelectron microscopy. Methods Mol. Biol. 117, 31-48 (1999).
71. Chen, B. et al. Structural dynamics of ribosome subunit association studied by mixing-spraying timeresolved cryogenic electron microscopy. Structure 23, 1097-1105 (2015).
72. Razinkov, I. et al. A new method for vitrifying samples for cryoEM. J. Struct. Biol. 195, 190-198 (2016).
73. Feng, X. et al. A fast and effective microfluidic spraying-plunging method for high-resolution singleparticle cryo-EM. Structure 25, 663-670 e663 (2017).
74. Berriman, J. &Unwin, N. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241-252 (1994).
75. Lu, Z. et al. Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy. J. Struct. Biol. 168, 388-395 (2009).
76. Shaikh, T. R. et al. Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM. Proc. Natl Acad. Sci. USA 111, 9822-9827 (2014).
77. Arnold, S. A. et al. Blotting-free and lossless cryoelectron microscopy grid preparation from nanolitersized protein samples and single-cell extracts. J. Struct. Biol. 197, 220-226 (2017).
78. Langmore, J. P. &Smith, M. F. Quantitative energyfiltered electron microscopy of biological molecules in ice. Ultramicroscopy 46, 349-373 (1992).
79. Orlova, E. V. &Saibil, H. R. Structural analysis of macromolecular assemblies by electron microscopy. Chem. Rev. 111, 7710-7748 (2011).
80. Bartesaghi, A. et al. 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cellpermeant inhibitor. Science 348, 1147-1151 (2015).
81. Glaeser, R. M. et al. Factors that influence the formation and stability of thin, cryo-EM specimens. Biophys. J. 110, 749-755 (2016).
82. Glaeser, R. M. &Han, B. G. Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys. Rep. 3, 1-7 (2017).
83. Russo, C. J. &Passmore, L. A. Progress towards an optimal specimen support for electron cryomicroscopy. Curr. Opin. Struct. Biol. 37, 81-89 (2016).
84. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793-796 (2017).
85. Trinick, J. &Cooper, J. Concentration of solutes during preparation of aqueous suspensions for cryo-electron microscopy. J. Microsc. 159, 215-222 (1990).
86. Egelhaaf, S. U., Schurtenberger, P. &Muller, M. New controlled environment vitrification system for cryotransmission electron microscopy: design and application to surfactant solutions. J. Microsc. 200, 128-139 (2000).
87. Ge, H., Suszynski, W. J., Davis, H. T. &Scriven, L. E. New controlled environment vitrification system for preparing wet samples for cryo-SEM. J. Microsc. 229, 115-126 (2008).
88. Iancu, C. V. et al. Electron cryotomography sample preparation using the Vitrobot. Nat. Protoc. 1, 2813-2819 (2006).
89. Resch, G. P., Brandstetter, M., Konigsmaier, L., Urban, E. &Pickl-Herk, A. M. Immersion freezing of suspended particles and cells for cryo-electron microscopy. Cold Spring Harb. Protoc. 2011, 803-814 (2011).
90. Kasas, S., Dumas, G., Dietler, G., Catsicas, S. &Adrian, M. Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging. J. Microsc. 211, 48-53 (2003).
91. Noble, A. J. et al. Routine single particle CryoEM sample and grid characterization by tomography. bioRxiv https://doi.org/10.1101/230276 (2017).
92. Wang, R. Y. et al. De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nat. Methods 12, 335-338 (2015).
93. Zhou, N., Wang, H. &Wang, J. EMBuilder: a template matching-based automatic model-building program for high-resolution Cryo-electron microscopy maps. Sci. Rep. 7, 2664 (2017).
94. Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).
95. Singharoy, A. et al. Molecular dynamics-based refinement and validation for sub-5 A cryo-electron microscopy maps. eLife 5, e16105 (2016).
96. Jakobi, A. J., Wilmanns, M. &Sachse, C. Model-based local density sharpening of cryo-EM maps. eLife 6, e27131 (2017).
97. Herzik, M. A. Jr., Wu, M. &Lander, G. C. Achieving better-than-3-A resolution by single-particle cryo-EM at 200 keV. Nat. Methods 14, 1075-1078 (2017).
98. Bai, X. C. et al. An atomic structure of human gammasecretase. Nature 525, 212-217 (2015).
99. Liao, M., Cao, E., Julius, D. &Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107-112 (2013).
100. Cao, E., Liao, M., Cheng, Y. &Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113-118 (2013).
101. Gao, Y., Cao, E., Julius, D. &Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347-351 (2016).
102. Paulsen, C. E., Armache, J. P., Gao, Y., Cheng, Y. &Julius, D. Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 520, 511-517 (2015).
103. Wu, J. et al. Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 A resolution. Nature 537, 191-196 (2016).
104. Shen, H. et al. Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution. Science 355, eaal4326 (2017).
105. Du, J., Lu, W., Wu, S., Cheng, Y. &Gouaux, E. Glycine receptor mechanism elucidated by electron cryomicroscopy. Nature 526, 224-229 (2015).
106. Hirschi, M. et al. Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550, 411-414 (2017).
107. Guo, J. et al. Structures of the calcium-activated, nonselective cation channel TRPM4. Nature 552, 205-209 (2017).
108. Ge, J. et al. Architecture of the mammalian mechanosensitive Piezo1 channel. Nature 527, 64-69 (2015).
109. Yan, Z. et al. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution. Nature 517, 50-55 (2015).
110. des Georges, A. et al. Structural basis for gating and activation of RyR1. Cell 167, 145-157 e117 (2016).
111. Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118-123 (2017).
112. Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248-253 (2017).
113. Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121-125 (2018).
114. García-Nafría, J., Lee, Y., Bai, X., Carpenter, B. &Tate, C. G. Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein. bioRxiv https://doi.org/10.1101/267674 (2018).
115. Carpenter, B., Nehme, R., Warne, T., Leslie, A. G. &Tate, C. G. Structure of the adenosine A(2A) receptor bound to an engineered G protein. Nature 536, 104-107 (2016).
116. Johnson, Z. L. &Chen, J. Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 168, 1075-1085 e1079 (2017).
117. Fitzpatrick, A. W. P. et al. Cryo-EM structures of tau filaments from Alzheimer's disease. Nature 547, 185-190 (2017).
118. Gremer, L. et al. Fibril structure of amyloid-beta(1-42) by cryo-electron microscopy. Science 358, 116-119 (2017).
119. Kellogg, E. H. et al. Insights into the distinct mechanisms of action of taxane and non-taxane microtubule stabilizers from cryo-EM structures. J. Mol. Biol. 429, 633-646 (2017).
120. Long, F. et al. Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity. Proc. Natl Acad. Sci. USA 112, 13898-13903 (2015).
121. Ciferri, C. et al. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody. Biochem. J. 472, 169-181 (2015).
122. Lee, J. H., Ozorowski, G. &Ward, A. B. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer. Science 351, 1043-1048 (2016).
123. Liu, Q. et al. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer. Nat. Struct. Mol. Biol. 24, 370-378 (2017).
124. Wong, W. et al. Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine. eLife 3, e03080 (2014).
125. Wong, W. et al. Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis. Nat. Microbiol. 2, 17031 (2017).
126. Khoshouei, M. et al. Volta phase plate cryo-EM of the small protein complex Prx3. Nat. Commun. 7, 10534 (2016).
127. Frank, J. Advances in the field of single-particle cryoelectron microscopy over the last decade. Nat. Protoc. 12, 209-212 (2017).
128. Rubinstein, J. L. Cryo-EM captures the dynamics of ion channel opening. Cell 168, 341-343 (2017).
129. Stuart, D. I., Subramaniam, S. &Abrescia, N. G. The democratization of cryo-EM. Nat. Methods 13, 607-608 (2016).
130. Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19-34 (2017).
131. Hauer, F. et al. GraDeR: membrane protein complex preparation for single-particle cryo-EM. Structure 23, 1769-1775 (2015).
132. Tribet, C., Audebert, R. &Popot, J. L. Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc. Natl Acad. Sci. USA 93, 15047-15050 (1996).
133. Nath, A., Atkins, W. M. &Sligar, S. G. Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins. Biochemistry 46, 2059-2069 (2007).
134. Tao, H. et al. Engineered nanostructured beta-sheet peptides protect membrane proteins. Nat. Methods 10, 759-761 (2013).
135. Scheres, S. H. et al. Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat. Methods 4, 27-29 (2007).
136. Lyumkis, D., Brilot, A. F., Theobald, D. L. &Grigorieff, N. Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377-388 (2013).
137. Zhou, A. et al. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. eLife 4, e10180 (2015).
138. Taylor, N. M. I. et al. Structure of the human multidrug transporter ABCG2. Nature 546, 504-509 (2017).
139. Oldham, M. L. et al. A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter. Nature 529, 537-540 (2016).
140. Karplus, P. A. &Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030-1033 (2012).
141. van Heel, M. &Schatz, M. Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250-262 (2005).
142. Neumann, P., Dickmanns, A. &Ficner, R. Validating resolution revolution. Structure https://doi. org/10.1016/j.str.2018.03.004 (2018).
143. Penczek, P. A., Grassucci, R. A. &Frank, J. The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryoelectron microscopy of biological particles. Ultramicroscopy 53, 251-270 (1994).
144. Sigworth, F. J. A maximum-likelihood approach to single-particle image refinement. J. Struct. Biol. 122, 328-339 (1998).
145. Milazzo, A. C. et al. Initial evaluation of a direct detection device detector for single particle cryoelectron microscopy. J. Struct. Biol. 176, 404-408 (2011).
146. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584-590 (2013).
147. Veesler, D. et al. Maximizing the potential of electron cryomicroscopy data collected using direct detectors. J. Struct. Biol. 184, 193-202 (2013).
148. McMullan, G., Faruqi, A. R., Clare, D. &Henderson, R. Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147, 156-163 (2014).
149. Kuijper, M. et al. FEI's direct electron detector developments: embarking on a revolution in cryo-TEM. J. Struct. Biol. 192, 179-187 (2015).
150. De Rosier, D. J. &Klug, A. Reconstruction of three dimensional structures from electron micrographs. Nature 217, 130-134 (1968).
151. Matadeen, R. et al. The Escherichia coli large ribosomal subunit at 7.5 A resolution. Structure 7, 1575-1583 (1999).
152. Ludtke, S. J., Chen, D. H., Song, J. L., Chuang, D. T. &Chiu, W. Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy. Structure 12, 1129-1136 (2004).
153. Zhang, X. et al. Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proc. Natl Acad. Sci. USA 105, 1867-1872 (2008).
154. Bai, X. C., Fernandez, I. S., McMullan, G. &Scheres, S. H. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013).
155. Fernandez, I. S. et al. Molecular architecture of a eukaryotic translational initiation complex. Science 342, 1240585 (2013).
156. Fischer, N. et al. Structure of the E. coli ribosome-EF-Tu complex at 3 A resolution by Cs-corrected cryo-EM. Nature 520, 567-570 (2015).
157. Jiang, J., Pentelute, B. L., Collier, R. J. &Zhou, Z. H. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521, 545-549 (2015).
158. Campbell, M. G., Veesler, D., Cheng, A., Potter, C. S. &Carragher, B. 2.8 A resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy. eLife 4, e06380 (2015).
159. Eisenberg, D. Max Perutz's achievements: how did he do it? Protein Sci. 3, 1625-1628 (1994).
160. Kendrew, J. C. et al. A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature 181, 662-666 (1958).
161. Kendrew, J. C. et al. Structure of myoglobin: a threedimensional fourier synthesis at 2 A. resolution. Nature 185, 422-427 (1960).
162. Perutz, M. F. et al. Structure of haemoglobin: a threedimensional fourier synthesis at 5.5-A. resolution, obtained by X-ray analysis. Nature 185, 416-422 (1960).
163. Adams, M. J. et al. Structure of rhombohedral 2 zinc insulin crystals. Nature 224, 491 (1969).
164. Protein Data Bank. Protein Data Bank. Nat. New Biol. 233, 223 (1971).
165. Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763 (1994).
166. Ondetti, M. A., Rubin, B. &Cushman, D. W. Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science 196, 441-444 (1977).
167. Deisenhofer, J., Epp, O., Miki, K., Huber, R. &Michel, H. Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3A resolution. Nature 318, 618-624 (1985).
168. Miller, M., Jaskolski, M., Rao, J. K., Leis, J. &Wlodawer, A. Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature 337, 576-579 (1989).
169. Navia, M. A. et al. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature 337, 615-620 (1989).
170. Lapatto, R. et al. X-Ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature 342, 299-302 (1989).
171. Wlodawer, A. et al. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 245, 616-621 (1989).
172. Miller, M. et al. Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution. Science 246, 1149-1152 (1989).
173. Abrahams, J. P., Leslie, A. G., Lutter, R. &Walker, J. E. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621-628 (1994).
174. Baldwin, J. J. et al. Thienothiopyran-2-sulfonamides: novel topically active carbonic anhydrase inhibitors for the treatment of glaucoma. J. Med. Chem. 32, 2510-2513 (1989).
175. Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69-77 (1998).
176. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739-745 (2000).
177. Abola, E., Kuhn, P., Earnest, T. &Stevens, R. C. Automation of X-ray crystallography. Nat. Struct. Biol. 7, S973-S977 (2000).
178. Muchmore, S. W. et al. Automated crystal mounting and data collection for protein crystallography. Structure 8, R243-R246 (2000).
179. Cohen, A. E., Ellis, P. J., Miller, M. D., Deacon, A. M. &Phizackerley, R. P. An automated system to mount cryo-cooled protein crystals on a synchrotron beam line, using compact sample cassettes and a small-scale robot. J. Appl. Crystallogr. 35, 720-726 (2002).
180. Beteva, A. et al. High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline. Acta Crystallogr. D Biol. Crystallogr. 62, 1162-1169 (2006).
181. Cipriani, F. et al. Automation of sample mounting for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 62, 1251-1259 (2006).
182. Hardy, L. W. &Malikayil, A. The impact of structureguided drug design on clinical agents. Curr. Drug Discov. 11, 15-20 (2003).
183. Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524-1529 (2011).
184. Rasmussen, S. G. et al. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477, 549-555 (2011).
185. Bollag, G. et al. Vemurafenib: the first drug approved for BRAF-mutant cancer. Nat. Rev. Drug Discov. 11, 873-886 (2012).
186. Erlanson, D. A., Fesik, S. W., Hubbard, R. E., Jahnke, W. &Jhoti, H. Twenty years on: the impact of fragments on drug discovery. Nat. Rev. Drug Discov. 15, 605-619 (2016).
187. Purcell, E. M., Torrey, H. C. &Pound, R. V. Resonance absorption by nuclear magnetic moments in a solid. Phys. Rev. 69, 37-38 (1946).
188. Bloch, F., Hansen, W. W. &Packard, M. Nuclear Induction. Phys. Rev. 69, 127 (1946).
189. Saunders, M., Wishnia, A. &Kirkwood, J. G. The nuclear magnetic resonance spectrum of ribonuclease. J. Am. Chem. Soc. 79, 3289-3290 (1957).
190. Ernst, R. R. Sensitivity enhancement in magnetic resonance. Adv. Magn. Reson. 2, 1-135 (1966).
191. Ernst, R. R. &Anderson, W. A. Application of fourier transform spectroscopy to magnetic resonance. Rev. Sci. Instrum. 37, 93 (1966).
192. Jeener, J. Oral presentation. Ampere International Summer School (Basko Polje, 1971).
193. Aue, W. P., Bartholdi, E. &Ernst, R. R. Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229-2246 (1976).
194. Bax, A. &Grzesiek, S. Methodological advances in protein NMR. Acc. Chem. Res. 26, 131-138 (1993).
195. Williamson, M. P., Havel, T. F. &Wuthrich, K. Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry. J. Mol. Biol. 182, 295-315 (1985).
196. Oschkinat, H. et al. Three-dimensional NMR spectroscopy of a protein in solution. Nature 332, 374-376 (1988).
197. Vuister, G. W., Boelens, R. &Kaptein, R. Nonselective three-dimensional NMR spectroscopy. The 3D NOEHOHAHA experiment. J. Magn. Reson. 80, 176-185 (1988).
198. Shuker, S. B., Hajduk, P. J., Meadows, R. P. &Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531-1534 (1996).
199. Mayer, M. &Meyer, B. Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew. Chem. Int. Ed 38, 1784-1788 (1999).
200. Hajduk, P. J. et al. NMR-based screening of proteins containing 13C-labeled methyl groups. J. Am. Chem. Soc. 122, 7898-7904 (2000).
201. Dalvit, C. et al. Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J. Biomol. NMR 18, 65-68 (2000).
202. Jahnke, W. Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy. Chembiochem 3, 167-173 (2002).
203. Jahnke, W. et al. NMR reporter screening for the detection of high-affinity ligands. Angew. Chem. Int. Ed Engl. 41, 3420-3423 (2002).
204. Dalvit, C., Fagerness, P. E., Hadden, D. T., Sarver, R. W. &Stockman, B. J. Fluorine-NMR experiments for highthroughput screening: theoretical aspects, practical considerations, and range of applicability. J. Am. Chem. Soc. 125, 7696-7703 (2003).
205. Ludwiczek, M. L., Baminger, B. &Konrat, R. NMR probing of protein-protein interactions using reporter ligands and affinity tags. J. Am. Chem. Soc. 126, 1636-1637 (2004).
206. Bruker. Bruker Introduces SampleXpress(TM), a New and Easy-to-Use Autosampler that Delivers Increased Efficiency for NMR. Bruker http://ir.bruker.com/ investors/press-releases/press-release-details/2010/ Bruker-Introduces-SampleXpressTM-A-New-and-Easyto-Use-Autosampler-that-Delivers-Increased-Efficiencyfor-NMR/default.aspx (2010).
207. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129-228 (1988).
208. Henderson, R. et al. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929 (1990).
209. Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171-193 (1995).
210. Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M. &Henderson, R. Electroncrystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259, 393-421 (1996).
211. Tagari, M., Newman, R., Chagoyen, M., Carazo, J. M. &Henrick, K. New electron microscopy database and deposition system. Trends Biochem. Sci. 27, 589 (2002).
212. Gonen, T. et al. Lipid-protein interactions in doublelayered two-dimensional AQP0 crystals. Nature 438, 633-638 (2005).
213. Jiang, W. et al. Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 451, 1130-1134 (2008).
214. Yu, X., Jin, L. &Zhou, Z. H. 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453, 415-419 (2008).
215. Community Research and Development Information Service. High-throughput three-dimensional electron microscopy. CORDIS https://cordis.europa.eu/project/ rcn/78402en.html (2009).
216. Ludtke, S. J. et al. De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure 16, 441-448 (2008).
217. Wood, C. et al. Collaborative computational project for electron cryo-microscopy. Acta Crystallogr. D Biol. Crystallogr. 71, 123-126 (2015).
218. Beck, F. et al. Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl Acad. Sci. USA 109, 14870-14875 (2012).
219. Allegretti, M., Mills, D. J., McMullan, G., Kuhlbrandt, W. &Vonck, J. Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryomicroscopy using a direct electron detector. eLife 3, e01963 (2014).
220. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485-1489 (2014).
221. Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353-358 (2016).
222. He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359-365 (2016).
223. Tsai, K. L. et al. Mediator structure and rearrangements required for holoenzyme formation. Nature 544, 196-201 (2017).
224. Schilbach, S. et al. Structures of transcription preinitiation complex with TFIIH and Mediator. Nature 551, 204-209 (2017).
225. Nguyen, T. H. D. et al. Cryo-EM structure of the yeast U4/U6. U5 tri-snRNP at 3.7 A resolution. Nature 530, 298-302 (2016).
226. Agafonov, D. E. et al. Molecular architecture of the human U4/U6. U5 tri-snRNP. Science 351, 1416-1420 (2016).
227. Yan, C. et al. Structure of a yeast spliceosome at 3.6-angstrom resolution. Science 349, 1182-1191 (2015).
228. Plaschka, C., Lin, P. C. &Nagai, K. Structure of a precatalytic spliceosome. Nature 546, 617-621 (2017).
229. Yan, C., Wan, R., Bai, R., Huang, G. &Shi, Y. Structure of a yeast activated spliceosome at 3.5 A resolution. Science 353, 904-911 (2016).
230. Rauhut, R. et al. Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science 353, 1399-1405 (2016).
231. Galej, W. P. et al. Cryo-EM structure of the spliceosome immediately after branching. Nature 537, 197-201 (2016).
232. Wan, R., Yan, C., Bai, R., Huang, G. &Shi, Y. Structure of a yeast catalytic step I spliceosome at 3.4 A resolution. Science 353, 895-904 (2016).
233. Zhang, X. et al. An atomic structure of the human spliceosome. Cell 169, 918-929 e914 (2017).
234. Bertram, K. et al. Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Nature 542, 318-323 (2017).
235. Yan, C., Wan, R., Bai, R., Huang, G. &Shi, Y. Structure of a yeast step II catalytically activated spliceosome. Science 355, 149-155 (2017).
236. Fica, S. M. et al. Structure of a spliceosome remodelled for exon ligation. Nature 542, 377-380 (2017).
237. Wilkinson, M. E. et al. Postcatalytic spliceosome structure reveals mechanism of 3?-splice site selection. Science 358, 1283-1288 (2017).
238. Liu, S. et al. Structure of the yeast spliceosomal postcatalytic P complex. Science 358, 1278-1283 (2017).
239. Sirohi, D. et al. The 3.8 A resolution cryo-EM structure of Zika virus. Science 352, 467-470 (2016).
240. Yuan, S. et al. Cryo-EM structure of a herpesvirus capsid at 3.1 A. Science 360, eaao7283 (2018).
241. von der Ecken, J., Heissler, S. M., Pathan-Chhatbar, S., Manstein, D. J. &Raunser, S. Cryo-EM structure of a human cytoplasmic actomyosin complex at nearatomic resolution. Nature 534, 724-728 (2016).
242. Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358-361 (2016).
243. Wu, M., Gu, J., Guo, R., Huang, Y. &Yang, M. Structure of mammalian respiratory supercomplex I1III2IV1. Cell 167, 1598-1609 e1510 (2016).
244. Guo, R., Zong, S., Wu, M., Gu, J. &Yang, M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2. Cell 170, 1247-1257 e1212 (2017).
245. Gristick, H. B. et al. Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site. Nat. Struct. Mol. Biol. 23, 906-915 (2016).
246. Dambacher, C. M., Worden, E. J., Herzik, M. A., Martin, A. &Lander, G. C. Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition. eLife 5, e13027 (2016).
247. Luan, B. et al. Structure of an endogenous yeast 26S proteasome reveals two major conformational states. Proc. Natl Acad. Sci. USA 113, 2642-2647 (2016).
248. Huang, X., Luan, B., Wu, J. &Shi, Y. An atomic structure of the human 26S proteasome. Nat. Struct. Mol. Biol. 23, 778-785 (2016).
249. Fischer, N. et al. The pathway to GTPase activation of elongation factor SelB on the ribosome. Nature 540, 80-85 (2016).
250. Natchiar, S. K., Myasnikov, A. G., Kratzat, H., Hazemann, I. &Klaholz, B. P. Visualization of chemical modifications in the human 80S ribosome structure. Nature 551, 472-477 (2017).
251. Barandun, J. et al. The complete structure of the smallsubunit processome. Nat. Struct. Mol. Biol. 24, 944-953 (2017).
252. Cheng, J., Kellner, N., Berninghausen, O., Hurt, E. &Beckmann, R. 3.2-A-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage. Nat. Struct. Mol. Biol. 24, 954-964 (2017).
253. Sanghai, Z. A. et al. Modular assembly of the nucleolar pre-60S ribosomal subunit. Nature 556, 126-129 (2018).
254. Zhang, R., Alushin, G. M., Brown, A. &Nogales, E. Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell 162, 849-859 (2015).
255. Passos, D. O. et al. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355, 89-92 (2017).
256. Bednar, J. et al. Structure and dynamics of a 197 bp nucleosome in complex with linker histone H1. Mol. Cell 66, 384-397.e8 (2017).
257. Ilangovan, A. et al. Cryo-EM structure of a relaxase reveals the molecular basis of DNA unwinding during bacterial conjugation. Cell 169, 708-721.e12 (2017).
258. Chowdhury, S. et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell 169, 47-57.e11 (2017).
259. Guo, T. W. et al. Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Cell 171, 414-426.e12 (2017).
260. Brown, A. et al. Structures of the human mitochondrial ribosome in native states of assembly. Nat. Struct. Mol. Biol. 24, 866-869 (2017).
261. Casanal, A. et al. Architecture of eukaryotic mRNA 3'-end processing machinery. Science 358, 1056-1059 (2017).
262. Clerici, M., Faini, M., Muckenfuss, L. M., Aebersold, R. &Jinek, M. Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex. Nat. Struct. Mol. Biol. 25, 135-138 (2018).
263. Urnavicius, L. et al. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature 554, 202-206 (2018).
264. Poepsel, S., Kasinath, V. &Nogales, E. Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes. Nat. Struct. Mol. Biol. 25, 154-162 (2018).
265. Bai, L., Wang, T., Zhao, G., Kovach, A. &Li, H. The atomic structure of a eukaryotic oligosaccharyltransferase complex. Nature 555, 328-333 (2018).
266. Shen, K. et al. Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes. Nature 556, 64-69 (2018).
267. Zyryanova, A. F. et al. Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B. Science 359, 1533-1536 (2018).
268. Scapin, G. et al. Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis. Nature 556, 122-125 (2018).
269. Meyerson, J. R. et al. Structural basis of kainate subtype glutamate receptor desensitization. Nature 537, 567-571 (2016).
270. Matthies, D. et al. Cryo-EM structures of the magnesium channel CorA reveal symmetry break upon gating. Cell 164, 747-756 (2016).
271. Whicher, J. R. &MacKinnon, R. Structure of the voltage-gated K(+) channel Eag1 reveals an alternative voltage sensing mechanism. Science 353, 664-669 (2016).
272. Tao, X., Hite, R. K. &MacKinnon, R. Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature 541, 46-51 (2017).
273. Li, M. et al. Structure of a eukaryotic cyclic-nucleotidegated channel. Nature 542, 60-65 (2017).
274. Lee, C. H. &MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111-120 e111 (2017).
275. Basak, S. et al. Cryo-EM structure of 5-HT3A receptor in its resting conformation. Nat. Commun. 9, 514 (2018).
276. Zhang, Z. &Chen, J. Atomic structure of the cystic fibrosis transmembrane conductance regulator. Cell 167, 1586-1597 e1589 (2016).
277. Lu, W., Du, J., Goehring, A. &Gouaux, E. Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation. Science 355, eaal3729 (2017).
278. Wang, W. &MacKinnon, R. Cryo-EM structure of the open human ether-A-go-go-related K+ channel hERG. Cell 169, 422-430 e410 (2017).
279. Jackson, S. M. et al. Structural basis of small-molecule inhibition of human multidrug transporter ABCG2. Nat. Struct. Mol. Biol. 25, 333-340 (2018).
280. Schmiege, P., Fine, M., Blobel, G. &Li, X. Human TRPML1 channel structures in open and closed conformations. Nature 550, 366-370 (2017).
281. Chen, Q. et al. Structure of mammalian endolysosomal TRPML1 channel in nanodiscs. Nature 550, 415-418 (2017).
282. Winkler, P. A., Huang, Y., Sun, W., Du, J. &Lu, W. Electron cryo-microscopy structure of a human TRPM4 channel. Nature 552, 200-204 (2017).
283. Martin, G. M., Kandasamy, B., DiMaio, F., Yoshioka, C. &Shyng, S. L. Anti-diabetic drug binding site in a mammalian KATP channel revealed by Cryo-EM. eLife 6, e31054 (2017).
284. Zubcevic, L. et al. Cryo-electron microscopy structure of the TRPV2 ion channel. Nat. Struct. Mol. Biol. 23, 180-186 (2016).
285. Deng, Z. et al. Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms. Nat. Struct. Mol. Biol. 25, 252-260 (2018).
286. Hughes, T. E. T. et al. Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat. Struct. Mol. Biol. 25, 53-60 (2018).
287. Shen, P. S. et al. The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs. Cell 167, 763-773 e711 (2016).
288. Roh, S. H. et al. The 3.5-A CryoEM structure of nanodisc-reconstituted yeast vacuolar ATPase Vo proton channel. Mol. Cell 69, 993-1004 e1003 (2018).
289. She, J. et al. Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel. Nature 556, 130-134 (2018).
290. Qian, P., Siebert, C. A., Wang, P., Canniffe, D. P. &Hunter, C. N. Cryo-EM structure of the Blastochloris viridis LH1-RC complex at 2.9 Å. Nature 556, 203-208 (2018).
291. Sun, C. et al. Structure of the alternative complex III in a supercomplex with cytochrome oxidase. Nature 557, 123-126 (2018).
292. Ruan, J., Xia, S., Liu, X., Lieberman, J. &Wu, H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature 557, 62-67 (2018).
293. Chua, E. Y. et al. 3.9 A structure of the nucleosome core particle determined by phase-plate cryo-EM. Nucleic Acids Res. 44, 8013-8019 (2016).
294. Xie, Q. et al. The 2.8 A electron microscopy structure of adeno-associated virus-DJ bound by a heparinoid pentasaccharide. Mol. Ther. Methods Clin. Dev. 5, 1-12 (2017).
295. Shalev-Benami, M. et al. Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin. Nat. Commun. 8, 1589 (2017).
296. Liu, Z. et al. Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit. Proc. Natl Acad. Sci. USA 113, 12174-12179 (2016).
297. Liu, Z. et al. Determination of the ribosome structure to a resolution of 2.5 A by single-particle cryo-EM. Protein Sci. 26, 82-92 (2017).
298. Banerjee, S. et al. 2.3 A resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 351, 871-875 (2016).
299. Dong, Y. et al. Antibody-induced uncoating of human rhinovirus B14. Proc. Natl Acad. Sci. USA 114, 8017-8022 (2017).
300. Golas, M. M. et al. Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo. EMBO J. 28, 766-778 (2009).
301. Sander, B., Golas, M. M., Lührmann, R. &Stark, H. An approach for de novo structure determination of dynamic molecular assemblies by electron cryomicroscopy. Structure 18, 667-676 (2010).
302. Tsai, J. C. et al. Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule. Science 359, eaaq0939 (2018).