Unravelling biological macromolecules with cryo-electron microscopy

Abstract

Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.

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Figure 1: Growth in structural biology over the past 40 years.
Figure 2: Membrane protein structural biology.
Figure 3: Soluble macromolecular machines.
Figure 4: Image classification enables the study of macromolecular dynamics.

References

  1. 1

    Alberts, B. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294 (1998).

  2. 2

    Banerjee, S. et al. 2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition. Science 351, 871–875 (2016).

  3. 3

    Kühlbrandt, W. The resolution revolution. Science 343, 1443–1444 (2014).

  4. 4

    Taylor, K. A. & Glaeser, R. M. Electron diffraction of frozen, hydrated protein crystals. Science 186, 1036–1037 (1974).

  5. 5

    Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J. & McDowall, A. W. Frozen aqueous suspensions. Ultramicroscopy 10, 55–62 (1982).

  6. 6

    Frank, J., Verschoor, A. & Boublik, M. Computer averaging of electron micrographs of 40S ribosomal subunits. Science 214, 1353–1355 (1981).

  7. 7

    Jiang, W. et al. Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy. Nature 451, 1130–1134 (2008).

  8. 8

    Yu, X., Jin, L. & Zhou, Z. H. 3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453, 415–419 (2008).

  9. 9

    Zhang, X. et al. Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proc. Natl Acad. Sci. USA 105, 1867–1872 (2008).

  10. 10

    Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl Acad. Sci. USA 106, 1063–1068 (2009).

  11. 11

    Schuette, J.-C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).

  12. 12

    Seidelt, B. et al. Structural insight into nascent polypeptide chain-mediated translational stalling. Science 326, 1412–1415 (2009).

  13. 13

    Agard, D., Cheng, Y., Glaeser, R. M. & Subramaniam, S. in Advances in Imaging and Electron Physics Vol. 185 (ed. Hawkes, P. W.) Ch. 2, 113–137 (Elsevier, 2014).

  14. 14

    Frank, J. Generalized single-particle cryo-EM—a historical perspective. Microscopy 65, 3–8 (2016).

  15. 15

    Vinothkumar, K. R. & Henderson, R. Single particle electron cryomicroscopy: trends, issues and future perspective. Q. Rev. Biophys. 49, e13 (2016).

  16. 16

    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).

  17. 17

    Krivanek, O. L. & Mooney, P. E. Applications of slow-scan CCD cameras in transmission electron microscopy. Ultramicroscopy 49, 95–108 (1993).

  18. 18

    Potter, C. S. et al. Leginon: a system for fully automated acquisition of 1000 electron micrographs a day. Ultramicroscopy 77, 153–161 (1999).

  19. 19

    McMullan, G., Chen, S., Henderson, R. & Faruqi, A. R. Detective quantum efficiency of electron area detectors in electron microscopy. Ultramicroscopy 109, 1126–1143 (2009).

  20. 20

    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). A comparison of the three commercially available direct electron detectors; all were shown to perform better than photographic film.

  21. 21

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013).

  22. 22

    Brilot, A. F. et al. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177, 630–637 (2012).

  23. 23

    Campbell, M. G. et al. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20, 1823–1828 (2012).

  24. 24

    Bai, X.-C., Fernandez, I. S., McMullan, G. & Scheres, S. H. W. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013).

  25. 25

    Scheres, S. H. W. et al. Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nature Methods 4, 27–29 (2007).

  26. 26

    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).

  27. 27

    Sigworth, F. J. A. Maximum-likelihood approach to single-particle image refinement. J. Struct. Biol. 122, 328–339 (1998).

  28. 28

    Clare, D. K. et al. ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin. Cell 149, 113–123 (2012).

  29. 29

    Allegretti, M., Mills, D. J., McMullan, G., Kühlbrandt, W. & Vonck, J. Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector. eLife 3, e01963 (2014). Refs 29–31 each present one of the first three high-resolution structures that heralded the resolution revolution in cryo-EM-based structure determination.

  30. 30

    Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013).

  31. 31

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

  32. 32

    Scheres, S. H. W. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012). A statistical framework for the classification and high-resolution refinement of cryo-EM structures that reduces the need for expert supervision.

  33. 33

    Bai, X.-C. et al. An atomic structure of human γ-secretase. Nature 525, 212–217 (2015).

  34. 34

    Merk, A. et al. Breaking cryo-EM resolution barriers to facilitate drug discovery. Cell 165, 1698–1707 (2016). The highest reported resolution (1.8 Å) of a single-particle reconstruction, at present.

  35. 35

    Campbell, M. G., Veesler, D., Cheng, A., Potter, C. S. & Carragher, B. 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy. eLife 4, e06380 (2015).

  36. 36

    Bartesaghi, A. et al. 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science 348, 1147–1151 (2015).

  37. 37

    Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015).

  38. 38

    Althoff, T., Mills, D. J., Popot, J. L. & Kühlbrandt, W. Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1 . EMBO J. 30, 4652–4664 (2011).

  39. 39

    Bayburt, T. H. & Sligar, S. G. Membrane protein assembly into nanodiscs. FEBS Lett. 584, 1721–1727 (2010).

  40. 40

    Frauenfeld, J. et al. A saposin-lipoprotein nanoparticle system for membrane proteins. Nature Methods 13, 345–351 (2016).

  41. 41

    Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013).

  42. 42

    Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).

  43. 43

    Wu, J. et al. Structure of the voltage-gated calcium channel CaV1.1 complex. Science 350, aad2395 (2015).

  44. 44

    Hite, R. K. et al. Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel. Nature 527, 198–203 (2015).

  45. 45

    Du, J., Lü, W., Wu, S., Cheng, Y. & Gouaux, E. Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526, 224–229 (2015).

  46. 46

    Baker, M. Making membrane proteins for structures: a trillion tiny tweaks. Nature Methods 7, 429–434 (2010).

  47. 47

    Lyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science 342, 1484–1490 (2013).

  48. 48

    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).

  49. 49

    Misasi, J. et al. Structural and molecular basis for Ebola virus neutralization by protective human antibodies. Science 351, 1343–1346 (2016).

  50. 50

    Gong, X. et al. Structural insights into the Niemann–Pick C1 (NPC1)-mediated cholesterol transfer and Ebola infection. Cell 165, 1467–1478 (2016).

  51. 51

    Yan, Z. et al. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution. Nature 517, 50–55 (2015).

  52. 52

    Efremov, R. G., Leitner, A., Aebersold, R. & Raunser, S. Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517, 39–43 (2015).

  53. 53

    Zalk, R. et al. Structure of a mammalian ryanodine receptor. Nature 517, 44–49 (2015).

  54. 54

    Fan, G. et al. Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature 527, 336–341 (2015).

  55. 55

    Tajima, N. et al. Activation of NMDA receptors and the mechanism of inhibition by ifenprodil. Nature 534, 63–68 (2016).

  56. 56

    Zhu, S. et al. Mechanism of NMDA receptor inhibition and activation. Cell 165, 704–714 (2016).

  57. 57

    Herguedas, B. et al. Structure and organization of heteromeric AMPA-type glutamate receptors. Science 352, aad3873 (2016).

  58. 58

    Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature 514, 328–334 (2014).

  59. 59

    Vinothkumar, K. R., Zhu, J. & Hirst, J. Architecture of mammalian respiratory complex I. Nature 515, 80–84 (2014).

  60. 60

    Wei, X. et al. Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution. Nature 534, 69–74 (2016).

  61. 61

    Zhao, J., Benlekbir, S. & Rubinstein, J. L. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015). An example of how image classification can reveal numerous functional states of dynamic molecular machines from a single experiment.

  62. 62

    Allegretti, M. et al. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237–240 (2015).

  63. 63

    Amunts, A., Brown, A., Toots, J., Scheres, S. H. W. & Ramakrishnan, V. The structure of the human mitochondrial ribosome. Science 348, 95–98 (2015).

  64. 64

    Greber, B. J. et al. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348, 303–308 (2015).

  65. 65

    Zhang, L. et al. Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science 350, 404–409 (2015).

  66. 66

    Hu, Z. et al. Structural and biochemical basis for induced self-propagation of NLRC4. Science 350, 399–404 (2015).

  67. 67

    Nguyen, T. H. D. et al. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530, 298–302 (2016).

  68. 68

    Agafonov, D. E. et al. Molecular architecture of the human U4/U6.U5 tri-snRNP. Science 351, 1416–1420 (2016).

  69. 69

    Wan, R. et al. The 3.8 Å structure of the U4/U6.U5 tri-snRNP: insights into spliceosome assembly and catalysis. Science 351, 466–475 (2016).

  70. 70

    Mosadeghi, R. et al. Structural and kinetic analysis of the COP9–signalosome activation and the cullin–RING ubiquitin ligase deneddylation cycle. eLife 5, e12102 (2016).

  71. 71

    Cavadini, S. et al. Cullin–RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature 531, 598–603 (2016).

  72. 72

    Liu, J.-J. et al. CryoEM structure of yeast cytoplasmic exosome complex. Cell Res. 26, 822–837 (2016).

  73. 73

    Chang, L., Zhang, Z., Yang, J., McLaughlin, S. H. & Barford, D. Atomic structure of the APC/C and its mechanism of protein ubiquitination. Nature 522, 450–454 (2015).

  74. 74

    Zhang, S. et al. Molecular mechanism of APC/C activation by mitotic phosphorylation. Nature 533, 260–264 (2016).

  75. 75

    Schweitzer, A. et al. Structure of the human 26S proteasome at a resolution of 3.9 Å. Proc. Natl Acad. Sci. USA 113, 7816–7821 (2016).

  76. 76

    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).

  77. 77

    Zhou, Q. et al. Cryo-EM structure of SNAP-SNARE assembly in 20S particle. Cell Res. 25, 551–560 (2015).

  78. 78

    Zhao, M. et al. Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61–67 (2015).

  79. 79

    Chowdhury, S., Ketcham, S. A., Schroer, T. A. & Lander, G. C. Structural organization of the dynein–dynactin complex bound to microtubules. Nature Struct. Mol. Biol. 22, 345–347 (2015).

  80. 80

    Urnavicius, L. et al. The structure of the dynactin complex and its interaction with dynein. Science 347, 1441–1446 (2015).

  81. 81

    Verba, K. A. et al. Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352, 1542–1547 (2016).

  82. 82

    Aylett, C. H. S. et al. Architecture of human mTOR complex 1. Science 351, 48–52 (2016).

  83. 83

    Baretić, D., Berndt, A., Ohashi, Y., Johnson, C. M. & Williams, R. L. Tor forms a dimer through an N-terminal helical solenoid with a complex topology. Nature Commun. 7, 11016 (2016).

  84. 84

    Fernández-Leiro, R., Conrad, J., Scheres, S. H. & Lamers, M. H. Cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ. eLife 4, e11134 (2015).

  85. 85

    Li, N. et al. Structure of the eukaryotic MCM complex at 3.8 Å. Nature 524, 186–191 (2015).

  86. 86

    Yuan, Z. et al. Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation. Nature Struct. Mol. Biol. 23, 217–224 (2016).

  87. 87

    Abid Ali, F. et al. Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate. Nature Commun. 7, 10708 (2016).

  88. 88

    Hoffmann, N. A. et al. Molecular structures of unbound and transcribing RNA polymerase III. Nature 528, 231–236 (2015).

  89. 89

    Murakami, K. et al. Structure of an RNA polymerase II preinitiation complex. Proc. Natl Acad. Sci. USA 112, 13543–13548 (2015).

  90. 90

    Bernecky, C., Herzog, F., Baumeister, W., Plitzko, J. M. & Cramer, P. Structure of transcribing mammalian RNA polymerase II. Nature 529, 551–554 (2016).

  91. 91

    He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016).

  92. 92

    Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016).

  93. 93

    Zhang, X. et al. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus. Nature 527, 531–534 (2015).

  94. 94

    Liu, H. & Cheng, L. Cryo-EM shows the polymerase structures and a nonspooled genome within a dsRNA virus. Science 349, 1347–1350 (2015).

  95. 95

    Ru, H. et al. Molecular mechanism of V(D)J recombination from synaptic RAG1–RAG2 complex structures. Cell 163, 1138–1152 (2015); erratum 163, 1807 (2015).

  96. 96

    Maskell, D. P. et al. Structural basis for retroviral integration into nucleosomes. Nature 523, 366–369 (2015).

  97. 97

    Ballandras-Colas, A. et al. Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 530, 358–361 (2016).

  98. 98

    Qu, G. et al. Structure of a group II intron in complex with its reverse transcriptase. Nature Struct. Mol. Biol. 23, 549–557 (2016).

  99. 99

    Jiang, F. et al. Structures of a CRISPR–Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).

  100. 100

    Taylor, D. W. et al. Structures of the CRISPR–Cmr complex reveal mode of RNA target positioning. Science 348, 581–585 (2015).

  101. 101

    Behrmann, E. et al. Structural snapshots of actively translating human ribosomes. Cell 161, 845–857 (2015).

  102. 102

    Campbell, M. G. et al. Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200kV. J. Struct. Biol. 188, 183–187 (2014).

  103. 103

    Liang, B. et al. Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy. Cell 162, 314–327 (2015).

  104. 104

    Saibil, H. R., Grünewald, K. & Stuart, D. I. A national facility for biological cryo-electron microscopy. Acta Crystallogr. D 71, 127–135 (2015).

  105. 105

    Cianfrocco, M. A. & Leschziner, A. E. Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud. eLife 4, e06664 (2015).

  106. 106

    Schmeisser, M. et al. Parallel, distributed and GPU computing technologies in single-particle electron microscopy. Acta Crystallogr. D 65, 659–671 (2009).

  107. 107

    Kimanius, D., Forsberg, B. O., Scheres, S. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Preprint at http://dx.doi.org/10.1101/059717 (2016).

  108. 108

    McMullan, G., Clark, A. T., Turchetta, R. & Faruqi, A. R. Enhanced imaging in low dose electron microscopy using electron counting. Ultramicroscopy 109, 1411–1416 (2009).

  109. 109

    Danev, R. & Baumeister, W. Cryo-EM single particle analysis with the Volta phase plate. eLife 5, e13046 (2016).

  110. 110

    Fischer, N. et al. Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM. Nature 520, 567–570 (2015).

  111. 111

    Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

  112. 112

    Stark, H., Zemlin, F. & Boettcher, C. Electron radiation damage to protein crystals of bacteriorhodopsin at different temperatures. Ultramicroscopy 63, 75–79 (1996).

  113. 113

    Russo, C. J. & Passmore, L. A. Ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014).

  114. 114

    Russo, C. J. & Passmore, L. A. Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas. Nature Methods 11, 649–652 (2014).

  115. 115

    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).

  116. 116

    Kelly, D. F., Dukovski, D. & Walz, T. New applications for affinity grids in preparing EM specimens. Microsc. Microanal. 16 (suppl. S2), 840–841 (2010).

  117. 117

    Jain, T., Sheehan, P., Crum, J., Carragher, B. & Potter, C. S. Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012).

  118. 118

    Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 (1995).

  119. 119

    Chen, B. et al. Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Structure 23, 1097–1105 (2015).

  120. 120

    Dashti, A. et al. Trajectories of the ribosome as a Brownian nanomachine. Proc. Natl Acad. Sci. USA 111, 17492–17497 (2014).

  121. 121

    Li, H. et al. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016).

  122. 122

    Kosinski, J. et al. Molecular architecture of the inner ring scaffold of the human nuclear pore complex. Science 352, 363–365 (2016).

  123. 123

    Song, F. et al. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380 (2014).

  124. 124

    Baumeister, W. Electron tomography: towards visualizing the molecular organization of the cytoplasm. Curr. Opin. Struct. Biol. 12, 679–684 (2002).

  125. 125

    Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969–972 (2016). This work presents an exciting vision of how cryo-electron tomography and sub-tomogram averaging can bridge the gap between the structural biology of isolated complexes and cell biology.

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Acknowledgements

We thank Y. Cheng, D. Barford and R. Henderson for comments on an early version of this manuscript. S.H.W.S is funded by the UK Medical Research Council (MC_UP_A025_1013).

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Correspondence to Sjors H. W. Scheres.

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Fernandez-Leiro, R., Scheres, S. Unravelling biological macromolecules with cryo-electron microscopy. Nature 537, 339–346 (2016) doi:10.1038/nature19948

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