1932

Abstract

Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.

Loading

Article metrics loading...

/content/journals/10.1146/annurev-biophys-111622-091300
2023-05-09
2024-12-03
Loading full text...

Full text loading...

/deliver/fulltext/biophys/52/1/annurev-biophys-111622-091300.html?itemId=/content/journals/10.1146/annurev-biophys-111622-091300&mimeType=html&fmt=ahah

Literature Cited

  1. 1.
    Almo SC, Garforth SJ, Hillerich BS, Love JD, Seidel RD, Burley SK. 2013. Protein production from the structural genomics perspective: achievements and future needs. Curr. Opin. Struct. Biol. 23:335–44
    [Google Scholar]
  2. 2.
    Bai XC, McMullan G, Scheres SHW. 2015. How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40:49–57
    [Google Scholar]
  3. 3.
    Barford D, Takagi Y, Schultz P, Berger I. 2013. Baculovirus expression: tackling the complexity challenge. Curr. Opin. Struct. Biol. 23:357–64
    [Google Scholar]
  4. 4.
    Berger I, Fitzgerald DJ, Richmond TJ. 2004. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22:1583–87
    [Google Scholar]
  5. 5.
    Bottcher B, Wynne SA, Crowther RA. 1997. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386:88–91
    [Google Scholar]
  6. 6.
    Brilot AF, Chen JZ, Cheng AC, Pan JH, Harrison SC et al. 2012. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177:630–37
    [Google Scholar]
  7. 7.
    Bromberg R, Guo Y, Borek D, Otwinowski Z. 2019. High-resolution cryo-EM reconstructions in the presence of substantial aberrations. bioRxiv 798280. https://doi.org/10.1101/798280
  8. 8.
    Byrne B. 2015. Pichia pastoris as an expression host for membrane protein structural biology. Curr. Opin. Struct. Biol. 32:9–17
    [Google Scholar]
  9. 9.
    Campbell MG, Cheng A, Brilot AF, Moeller A, Lyumkis D et al. 2012. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20:1823–28
    [Google Scholar]
  10. 10.
    Castano-Diez D, Moser D, Schoenegger A, Pruggnaller S, Frangakis AS. 2008. Performance evaluation of image processing algorithms on the GPU. J. Struct. Biol. 164:153–60
    [Google Scholar]
  11. 11.
    Chari A, Fischer U. 2010. Cellular strategies for the assembly of molecular machines. Trends Biochem. Sci. 35:676–83
    [Google Scholar]
  12. 12.
    Chari A, Haselbach D, Kirves JM, Ohmer J, Paknia E et al. 2015. ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space. Nat. Methods 12:859–65
    [Google Scholar]
  13. 13.
    Chen J, Noble AJ, Kang JY, Darst SA. 2019. Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: bacterial RNA polymerase and CHAPSO. J. Struct. Biol. X 1:100005
    [Google Scholar]
  14. 14.
    Chen JZ, Settembre EC, Aoki ST, Zhang X, Bellamy AR et al. 2009. Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. PNAS 106:10644–48
    [Google Scholar]
  15. 15.
    Cheng YF. 2015. Single-particle cryo-EM at crystallographic resolution. Cell 161:450–57
    [Google Scholar]
  16. 16.
    Clough R, Kirkland AI. 2016. Direct digital electron detectors. Adv. Imaging Electron Phys. 198:1–42
    [Google Scholar]
  17. 17.
    D'Imprima E, Floris D, Joppe M, Sanchez R, Grininger M, Kuhlbrandt W 2019. Protein denaturation at the air-water interface and how to prevent it. eLife 8:e42747
    [Google Scholar]
  18. 18.
    Faruqi AR, Henderson R. 2007. Electronic detectors for electron microscopy. Curr. Opin. Struct. Biol. 17:549–55
    [Google Scholar]
  19. 19.
    Fischer N, Neumann P, Konevega AL, Bock LV, Ficner R et al. 2015. Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520:567–70
    [Google Scholar]
  20. 20.
    Forler D, Kocher T, Rode M, Gentzel M, Izaurralde E, Wilm M. 2003. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21:89–92
    [Google Scholar]
  21. 21.
    Frank J, Shimkin B, Dowse H. 1981. SPIDER—a modular software system for electron image processing. Ultramicroscopy 6:343–57
    [Google Scholar]
  22. 22.
    Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M et al. 2002. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–47
    [Google Scholar]
  23. 23.
    Glaeser RM. 2019. How good can single-particle cryo-EM become? What remains before it approaches its physical limits?. Annu. Rev. Biophys. 48:45–61
    [Google Scholar]
  24. 24.
    Glaeser RM, Han B-G. 2017. Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys. Rep. 3:1–7
    [Google Scholar]
  25. 25.
    Glaeser RM, Han B-G, Csencsits R, Killilea A, Pulk A, Cate JHD. 2016. Factors that influence the formation and stability of thin, cryo-EM specimens. Biophys. J. 110:749–55
    [Google Scholar]
  26. 26.
    Grant T, Grigorieff N 2015. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4:e06980
    [Google Scholar]
  27. 27.
    Grant T, Rohou A, Grigorieff N 2018. cisTEM, user-friendly software for single-particle image processing. eLife 7:e35383
    [Google Scholar]
  28. 28.
    Groll M, Kim KB, Kairies N, Huber R, Crews CM. 2000. Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of α′,β′-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 122:1237–38
    [Google Scholar]
  29. 29.
    Haider M, Muller H, Uhlemann S, Zach J, Loebau U, Hoeschen R. 2008. Prerequisites for a Cc/Cs-corrected ultrahigh-resolution TEM. Ultramicroscopy 108:167–78
    [Google Scholar]
  30. 30.
    Hakhverdyan Z, Domanski M, Hough LE, Oroskar AA, Oroskar AR et al. 2015. Rapid, optimized interactomic screening. Nat. Methods 12:553–60
    [Google Scholar]
  31. 31.
    Hamaguchi T, Maki-Yonekura S, Naitow H, Matsuura Y, Ishikawa T, Yonekura K. 2019. A new cryo-EM system for single particle analysis. J. Struct. Biol. 207:40–48
    [Google Scholar]
  32. 32.
    Harauz G, van Heel M. 1986. Exact filters for general geometry three dimensional reconstruction. Optik 73:146–56
    [Google Scholar]
  33. 33.
    Harshbarger W, Miller C, Diedrich C, Sacchettini J. 2015. Crystal structure of the human 20S proteasome in complex with carfilzomib. Structure 23:418–24
    [Google Scholar]
  34. 34.
    Hart DJ, Waldo GS. 2013. Library methods for structural biology of challenging proteins and their complexes. Curr. Opin. Struct. Biol. 23:403–8
    [Google Scholar]
  35. 35.
    Hayer-Hartl M, Hartl FU. 2020. Chaperone machineries of Rubisco—the most abundant enzyme. Trends Biochem. Sci. 45:748–63
    [Google Scholar]
  36. 36.
    Henderson R, Baldwin JM, Downing KH, Lepault J, Zemlin F. 1986. Structure of purple membrane from Halobacterium halobium—recording, measurement and evaluation of electron micrographs at 3.5 Å resolution. Ultramicroscopy 19:147–78
    [Google Scholar]
  37. 37.
    Henneberg F, Chari A 2021. Chromatography-free purification strategies for large biological macromolecular complexes involving fractionated PEG precipitation and density gradients. Life 11:1289
    [Google Scholar]
  38. 38.
    Hosokawa F, Sawada H, Kondo Y, Takayanagi K, Suenaga K. 2013. Development of Cs and Cc correctors for transmission electron microscopy. Microscopy 62:23–41
    [Google Scholar]
  39. 39.
    Jungbauer A, Machold C, Hahn R. 2005. Hydrophobic interaction chromatography of proteins—III. Unfolding of proteins upon adsorption. J. Chromatogr. A 1079:221–28
    [Google Scholar]
  40. 40.
    Kato T, Makino F, Nakane T, Terahara N, Kaneko T et al. 2019. The 1.54 Å resolution structure of apoferritin by CRYOARM300 with Cold-FEG. Microsc. Microanal. 2:Suppl.998–99
    [Google Scholar]
  41. 41.
    Kimanius D, Forsberg BO, Scheres SHW, Lindahl E 2016. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5:e18722
    [Google Scholar]
  42. 42.
    Kisselev AF. 2022. Site-specific proteasome inhibitors. Biomolecules 12:54
    [Google Scholar]
  43. 43.
    Klebl DP, Monteiro DCF, Kontziampasis D, Kopf F, Sobott F et al. 2020. Sample deposition onto cryo-EM grids: from sprays to jets and back. Acta Crystallogr. D 76:340–49
    [Google Scholar]
  44. 44.
    Kuhlbrandt W. 2014. The resolution revolution. Science 343:1443–44
    [Google Scholar]
  45. 45.
    Kuijper M, van Hoften G, Janssen B, Geurink R, De Carlo S et al. 2015. FEI's direct electron detector developments: embarking on a revolution in cryo-TEM. J. Struct. Biol. 192:179–87
    [Google Scholar]
  46. 46.
    LaCava J, Molloy KR, Taylor MS, Domanski M, Chait BT, Rout MP. 2015. Affinity proteomics to study endogenous protein complexes: pointers, pitfalls, preferences and perspectives. Biotechniques 58:103–19
    [Google Scholar]
  47. 47.
    Li XM, Grigorieff N, Cheng YF. 2010. GPU-enabled FREALIGN: accelerating single particle 3D reconstruction and refinement in Fourier space on graphics processors. J. Struct. Biol. 172:407–12
    [Google Scholar]
  48. 48.
    Li XM, Mooney P, Zheng S, Booth CR, Braunfeld MB et al. 2013. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10:584–90
    [Google Scholar]
  49. 49.
    Ludtke SJ, Baldwin PR, Chiu W. 1999. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128:82–97
    [Google Scholar]
  50. 50.
    Maeda S, Schertler GFX. 2013. Production of GPCR and GPCR complexes for structure determination. Curr. Opin. Struct. Biol. 23:381–92
    [Google Scholar]
  51. 51.
    Marabini R, Masegosa IM, San Martin MC, Marco S, Fernandez JJ et al. 1996. Xmipp: an image processing package for electron microscopy. J. Struct. Biol. 116:237–40
    [Google Scholar]
  52. 52.
    McMullan G, Faruqi AR, Clare D, Henderson R 2014. Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147:156–63
    [Google Scholar]
  53. 53.
    McMullan G, Faruqi AR, Henderson R. 2016. Direct electron detectors. Methods Enzymol 579:1–17
    [Google Scholar]
  54. 54.
    Mesa P, Deniaud A, Montoya G, Schaffitzel C. 2013. Directly from the source: endogenous preparations of molecular machines. Curr. Opin. Struct. Biol. 23:319–25
    [Google Scholar]
  55. 55.
    Milazzo AC, Cheng AC, Moeller A, Lyumkis D, Jacovetty E et al. 2011. Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy. J. Struct. Biol. 176:404–8
    [Google Scholar]
  56. 56.
    Moriya T, Saur M, Stabrin M, Merino F, Voicu H et al. 2017. High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE. J. Vis. Exp. 123:55448
    [Google Scholar]
  57. 57.
    Muller H, Massmann I, Uhlemann S, Hartel P, Zach J, Haider M 2011. Aplanatic imaging systems for the transmission electron microscope. Nucl. Instrum. Methods Phys. Res. A 645:20–27
    [Google Scholar]
  58. 58.
    Muller H, Uhlemann S, Hartel P, Haider M. 2008. Aberration-corrected optics: from an idea to a device. Proceedings of the 7th International Conference on Charged Particle Optics (CPO-7), Cambridge, United Kingdom, July 24–26, Vol. 1167–78. Red Hook, NY: Curran Assoc.
    [Google Scholar]
  59. 59.
    Nakane T, Kotecha A, Sente A, McMullan G, Masiulis S et al. 2020. Single-particle cryo-EM at atomic resolution. Nature 587:152–56
    [Google Scholar]
  60. 60.
    Naydenova K, Jia P, Russo CJ. 2020. Cryo-EM with sub-1 Å specimen movement. Science 370:223–26
    [Google Scholar]
  61. 61.
    Naydenova K, McMullan G, Peet MJ, Lee Y, Edwards PC et al. 2019. CryoEM at 100 keV: a demonstration and prospects. IUCrJ 6:1086–98
    [Google Scholar]
  62. 62.
    Noble AJ, Dandey VP, Wei H, Braschi J, Chase J et al. 2018. Routine single particle CryoEM sample and grid characterization by tomography. eLife 7:e34257
    [Google Scholar]
  63. 63.
    Peet MJ, Henderson R, Russo CJ. 2019. The energy dependence of contrast and damage in electron cryomicroscopy of biological molecules. Ultramicroscopy 203:125–31
    [Google Scholar]
  64. 64.
    Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA. 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14:290–96
    [Google Scholar]
  65. 65.
    Renaud JP, Chari A, Ciferri C, Liu WT, Remigy HW et al. 2018. Cryo-EM in drug discovery: achievements, limitations and prospects. Nat. Rev. Drug Discov. 17:471–92
    [Google Scholar]
  66. 66.
    Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17:1030–32
    [Google Scholar]
  67. 67.
    Rohou A, Grigorieff N. 2015. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192:216–21
    [Google Scholar]
  68. 68.
    Rosenthal PB, Henderson R. 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333:721–45
    [Google Scholar]
  69. 69.
    Russo CJ, Henderson R. 2018. Ewald sphere correction using a single side-band image processing algorithm. Ultramicroscopy 187:26–33
    [Google Scholar]
  70. 70.
    Scheres SHW. 2012. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180:519–30
    [Google Scholar]
  71. 71.
    Scherzer O. 1936. Over some errors of electron lenses. Z. Phys. 101:593–603
    [Google Scholar]
  72. 72.
    Schmeisser M, Heisen BC, Luettich M, Busche B, Hauer F et al. 2009. Parallel, distributed and GPU computing technologies in single-particle electron microscopy. Acta Crystallogr. D 65:659–71
    [Google Scholar]
  73. 73.
    Schrader J, Henneberg F, Mata RA, Tittmann K, Schneider TR et al. 2016. The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design. Science 353:594–98
    [Google Scholar]
  74. 74.
    Sigworth FJ. 1998. A maximum-likelihood approach to single-particle image refinement. J. Struct. Biol. 122:328–39
    [Google Scholar]
  75. 75.
    Singh K, Graf B, Linden A, Sautner V, Urlaub H et al. 2020. Discovery of a regulatory subunit of the yeast fatty acid synthase. Cell 180:1130–43.e20
    [Google Scholar]
  76. 76.
    Stark H. 2010. GraFix: stabilization of fragile macromolecular complexes for single particle cryo-EM. Methods Enzymol 481:109–26
    [Google Scholar]
  77. 77.
    Stark H, Chari A. 2016. Sample preparation of biological macromolecular assemblies for the determination of high-resolution structures by cryo-electron microscopy. Microscopy 65:23–34
    [Google Scholar]
  78. 78.
    Vanheel M, Keegstra W. 1981. IMAGIC—a fast, flexible and friendly image analysis software system. Ultramicroscopy 7:113–30
    [Google Scholar]
  79. 79.
    Vijayachandran LS, Viola C, Garzoni F, Trowitzsch S, Bieniossek C et al. 2011. Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J. Struct. Biol. 175:198–208
    [Google Scholar]
  80. 80.
    Vinothkumar KR, Henderson R. 2016. Single particle electron cryomicroscopy: trends, issues and future perspective. Q. Rev. Biophys. 49:e13
    [Google Scholar]
  81. 81.
    Yip KM, Fischer N, Paknia E, Chari A, Stark H. 2020. Atomic-resolution protein structure determination by cryo-EM. Nature 587:157–61
    [Google Scholar]
  82. 82.
    Yu X, Jin L, Zhou ZH 2008. 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453:415–19
    [Google Scholar]
  83. 83.
    Zemlin F, Beckmann E, van der Mast KD. 1996. A 200 kV electron microscope with Schottky field emitter and a helium-cooled superconducting objective lens. Ultramicroscopy 63:227–38
    [Google Scholar]
  84. 84.
    Zemlin F, Weiss K, Schiske P, Kunath W, Herrmann KH. 1978. Coma-free alignment of high resolution electron microscopes with the aid of optical diffractograms. Ultramicroscopy 3:49–60
    [Google Scholar]
  85. 85.
    Zhang K. 2016. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193:1–12
    [Google Scholar]
  86. 86.
    Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng YF, Agard DA. 2017. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14:331–32
    [Google Scholar]
  87. 87.
    Zhou ZH. 2011. Atomic resolution cryo electron microscopy of macromolecular complexes. Adv. Protein Chem. Struct. Biol. 82:1–35
    [Google Scholar]
  88. 88.
    Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJH et al. 2018. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7:e42166
    [Google Scholar]
  89. 89.
    Zivanov J, Nakane T, Scheres SHW. 2020. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7:253–67
    [Google Scholar]
/content/journals/10.1146/annurev-biophys-111622-091300
Loading
/content/journals/10.1146/annurev-biophys-111622-091300
Loading

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error