Single-Particle Cryo-EM of Membrane Proteins in Lipid Nanodiscs

  • Valeria Kalienkova
  • Carolina Alvadia
  • Vanessa Clerico Mosina
  • Cristina PaulinoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)


Single-particle cryo-electron microscopy has become an indispensable technique in structural biology. In particular when studying membrane proteins, it allows the use of membrane-mimicking tools, which can be crucial for a comprehensive understanding of the structure-function relationship of the protein in its native environment. In this chapter we focus on the application of nanodiscs and use our recent studies on the TMEM16 family as an example.

Key words

Cryo-EM Single-particle Nanodiscs Membrane proteins Structural biology 



We thank Hendrik Siekkema and Dawid Deneka for valuable discussion and Jan Rheinberger and Chancievan Thangaratnarajah for critical reading of the manuscript. V.K was funded by the SNF early postdocmobility fellowship 187679. C.P. was funded by the NWO Veni grant 722.017.001 and the NWO Start-Up grant 740.018.016.


  1. 1.
    Kühlbrandt W (2014) The resolution revolution. Science 343(6178):1443. Scholar
  2. 2.
    Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJH, Lindahl E, Scheres SHW (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7:e42166. Scholar
  3. 3.
    Fan X, Wang J, Zhang X, Yang Z, Zhang JC, Zhao L, Peng HL, Lei J, Wang HW (2019) Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution. Nat Commun 10(1):2386. Scholar
  4. 4.
    Roh SH, Stam NJ, Hryc CF, Couoh-Cardel S, Pintilie G, Chiu W, Wilkens S (2018) The 3.5-Å cryoEM structure of nanodisc-reconstituted yeast vacuolar ATPase Vo proton channel. Mol Cell 69(6):993–1004. Scholar
  5. 5.
    Matthies D, Bae C, Toombes GES, Fox T, Bartesaghi A, Subramaniam S, Swartz KJ (2018) Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs. Elife 7:e37558. Scholar
  6. 6.
    Autzen HE, Myasnikov AG, Campbell MG, Asarnow D, Julius D, Cheng Y (2018) Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359(6372):228–232. Scholar
  7. 7.
    Jojoa Cruz S, Saotome K, Murthy SE, Tsui CC, Sansom MSP, Patapoutian A, Ward AB (2018) Cryo-EM structure of the mechanically activated ion channel OSCA1.2. Elife 7:e41845. Scholar
  8. 8.
    Mi W, Li Y, Yoon SH, Ernst RK, Walz T, Liao M (2017) Structural basis of MsbA-mediated lipopolysaccharide transport. Nature 549:233–237. Scholar
  9. 9.
    Srivastava AP, Luo M, Zhou W, Symersky J, Bai D, Chambers MG, Faraldo-Gómez JD, Liao M, Mueller DM (2018) High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane. Science 360(6389):eaas9699CrossRefGoogle Scholar
  10. 10.
    Taylor NMI, Manolaridis I, Jackson SM, Kowal J, Stahlberg H, Locher KP (2017) Structure of the human multidrug transporter ABCG2. Nature 546:504–509. Scholar
  11. 11.
    Jackson SM, Manolaridis I, Kowal J, Zechner M, Taylor NMI, Bause M, Bauer S, Bartholomaeus R, Bernhardt G, Koenig B, Buschauer A, Stahlberg H, Altmann K-H, Locher KP (2018) Structural basis of small-molecule inhibition of human multidrug transporter ABCG2. Nat Struct Mol Biol 25(4):333–340. Scholar
  12. 12.
    Gatsogiannis C, Merino F, Prumbaum D, Roderer D, Leidreiter F, Meusch D, Raunser S (2016) Membrane insertion of a Tc toxin in near-atomic detail. Nat Struct Mol Biol 23:884–890. Scholar
  13. 13.
    Zhang S, Li N, Zeng W, Gao N, Yang M (2017) Cryo-EM structures of the mammalian endo-lysosomal TRPML1 channel elucidate the combined regulation mechanism. Protein Cell 8(11):834–847. Scholar
  14. 14.
    Chen Q, She J, Zeng W, Guo J, Xu H, Bai X-c, Jiang Y (2017) Structure of mammalian endolysosomal TRPML1 channel in nanodiscs. Nature 550:415. Scholar
  15. 15.
    Jin P, Bulkley D, Guo Y, Zhang W, Guo Z, Huynh W, Wu S, Meltzer S, Cheng T, Jan LY, Jan Y-N, Cheng Y (2017) Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547:118–122. Scholar
  16. 16.
    Gao Y, Cao E, Julius D, Cheng Y (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534:347–351. Scholar
  17. 17.
    Fan C, Fan M, Orlando BJ, Fastman NM, Zhang J, Xu Y, Chambers MG, Xu X, Perry K, Liao M, Feng L (2018) X-ray and cryo-EM structures of the mitochondrial calcium uniporter. Nature 559(7715):575–579. Scholar
  18. 18.
    Dang S, Feng S, Tien J, Peters CJ, Bulkley D, Lolicato M, Zhao J, Zuberbühler K, Ye W, Qi L, Chen T, Craik CS, Jan YN, Minor DL Jr, Cheng Y, Jan LY (2017) Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature 552:426–429. Scholar
  19. 19.
    Kalienkova V, Clerico Mosina V, Bryner L, Oostergetel GT, Dutzler R, Paulino C (2019) Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. Elife 8:e44364. Scholar
  20. 20.
    Falzone ME, Rheinberger J, Lee B-C, Peyear T, Sasset L, Raczkowski AM, Eng ET, Di Lorenzo A, Andersen OS, Nimigean CM, Accardi A (2019) Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase. Elife 8:e43229. Scholar
  21. 21.
    Alvadia C, Lim NK, Clerico Mosina V, Oostergetel GT, Dutzler R, Paulino C (2019) Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F. Elife 8:e44365. Scholar
  22. 22.
    Kern DM, Oh S, Hite RK, Brohawn SG (2019) Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs. Elife 8:e42636. Scholar
  23. 23.
    Tang Q, Guo W, Zheng L, Wu J-X, Liu M, Zhou X, Zhang X, Chen L (2018) Structure of the receptor-activated human TRPC6 and TRPC3 ion channels. Cell Res 28(7):746–755. Scholar
  24. 24.
    Hughes TET, Pumroy RA, Yazici AT, Kasimova MA, Fluck EC, Huynh KW, Samanta A, Molugu SK, Zhou ZH, Carnevale V, Rohacs T, Moiseenkova-Bell VY (2018) Structural insights on TRPV5 gating by endogenous modulators. Nat Commun 9(1):4198. Scholar
  25. 25.
    Shen PS, Yang X, DeCaen PG, Liu X, Bulkley D, Clapham DE, Cao E (2016) The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs. Cell 167(3):763–773. Scholar
  26. 26.
    Willegems K, Efremov RG (2018) Influence of lipid mimetics on gating of ryanodine receptor. Structure 26(10):1303–1313. Scholar
  27. 27.
    Wild R, Kowal J, Eyring J, Ngwa EM, Aebi M, Locher KP (2018) Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Science 359(6375):545–550. Scholar
  28. 28.
    McGoldrick LL, Singh AK, Saotome K, Yelshanskaya MV, Twomey EC, Grassucci RA, Sobolevsky AI (2017) Opening of the human epithelial calcium channel TRPV6. Nature 553:233–237. Scholar
  29. 29.
    Quentin D, Ahmad S, Shanthamoorthy P, Mougous JD, Whitney JC, Raunser S (2018) Mechanism of loading and translocation of type VI secretion system effector Tse6. Nat Microbiol 3(10):1142–1152. Scholar
  30. 30.
    Rheinberger J, Gao X, Schmidpeter PAM, Nimigean CM (2018) Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures. Elife 7:e39775. Scholar
  31. 31.
    Laverty D, Desai R, Uchański T, Masiulis S, Stec WJ, Malinauskas T, Zivanov J, Pardon E, Steyaert J, Miller KW, Aricescu AR (2019) Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer. Nature 565:516–520. Scholar
  32. 32.
    Saotome K, Teng B, Tsui CC, Lee W-H, Tu Y-H, Kaplan JP, Sansom MSP, Liman ER, Ward AB (2019) Structures of the otopetrin proton channels Otop1 and Otop3. Nat Struct Mol Biol 26(6):518–525. Scholar
  33. 33.
    Walter JD, Sawicka M, Dutzler R (2019) Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport. Elife 8:e46986. Scholar
  34. 34.
    Davies KM, Anselmi C, Wittig I, Faraldo-Gómez JD, Kühlbrandt W (2012) Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae. Proc Natl Acad Sci U S A 109(34):13602–13607. Scholar
  35. 35.
    Kosinski J, Mosalaganti S, von Appen A, Teimer R, DiGuilio AL, Wan W, Bui KH, Hagen WJH, Briggs JAG, Glavy JS, Hurt E, Beck M (2016) Molecular architecture of the inner ring scaffold of the human nuclear pore complex. Science 352(6283):363–365. Scholar
  36. 36.
    von Appen A, Kosinski J, Sparks L, Ori A, DiGuilio AL, Vollmer B, Mackmull M-T, Banterle N, Parca L, Kastritis P, Buczak K, Mosalaganti S, Hagen W, Andres-Pons A, Lemke EA, Bork P, Antonin W, Glavy JS, Bui KH, Beck M (2015) In situ structural analysis of the human nuclear pore complex. Nature 526:140–143. Scholar
  37. 37.
    Kovtun O, Leneva N, Bykov YS, Ariotti N, Teasdale RD, Schaffer M, Engel BD, Owen DJ, Briggs JAG, Collins BM (2018) Structure of the membrane-assembled retromer coat determined by cryo-electron tomography. Nature 561(7724):561–564. Scholar
  38. 38.
    Mühleip AW, Dewar CE, Schnaufer A, Kuhlbrandt W, Davies KM (2017) In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits. Proc Natl Acad Sci U S A 114(5):992–997. Scholar
  39. 39.
    Mühleip AW, Joos F, Wigge C, Frangakis AS, Kühlbrandt W, Davies KM (2016) Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria. Proc Natl Acad Sci U S A 113(30):8442–8447. Scholar
  40. 40.
    Tonggu L, Wang L (2018) Cryo-EM sample preparation method for extremely low concentration liposomes. bioRxiv:494997.
  41. 41.
    Kudryashev M, Castaño-Díez D, Deluz C, Hassaine G, Grasso L, Graf-Meyer A, Vogel H, Stahlberg H (2016) The structure of the mouse serotonin 5-HT3 receptor in lipid vesicles. Structure 24(1):165–170. Scholar
  42. 42.
    Pang SS, Bayly-Jones C, Radjainia M, Spicer BA, Law RHP, Hodel AW, Ekkel SM, Conroy PJ, Ramm G, Venugopal H, Bird PI, Hoogenboom BW, Voskoboinik I, Gambin Y, Sierecki E, Dunstone MA, Whisstock JC (2019) The structure of Macrophage Expressed Gene-1, a phagolysosome immune effector that is activated upon acidification. bioRxiv:580712.
  43. 43.
    Frauenfeld J, Löving R, Armache J-P, Sonnen AFP, Guettou F, Moberg P, Zhu L, Jegerschöld C, Flayhan A, Briggs JAG, Garoff H, Löw C, Cheng Y, Nordlund P (2016) A saposin-lipoprotein nanoparticle system for membrane proteins. Nat Methods 13:345–351. Scholar
  44. 44.
    Nagamura R, Fukuda M, Kawamoto A, Matoba K, Dohmae N, Ishitani R, Takagi J, Nureki O (2019) Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2. Acta Crystallogr F Struct Biol Commun 75(Pt 5):348–358. Scholar
  45. 45.
    Carlson ML, Young JW, Zhao Z, Fabre L, Jun D, Li J, Li J, Dhupar HS, Wason I, Mills AT, Beatty JT, Klassen JS, Rouiller I, Duong F (2018) The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution. Elife 7:e34085. Scholar
  46. 46.
    Knowles TJ, Finka R, Smith C, Lin Y-P, Dafforn T, Overduin M (2009) Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J Am Chem Soc 131(22):7484–7485. Scholar
  47. 47.
    Bayburt TH, Carlson JW, Sligar SG (1998) Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer. J Struct Biol 123(1):37–44. Scholar
  48. 48.
    Flayhan A, Mertens HDT, Ural-Blimke Y, Martinez Molledo M, Svergun DI, Löw C (2018) Saposin lipid nanoparticles: a highly versatile and modular tool for membrane protein research. Structure 26(2):345–355. Scholar
  49. 49.
    Nguyen NX, Armache J-P, Lee C, Yang Y, Zeng W, Mootha VK, Cheng Y, Bai X-c, Jiang Y (2018) Cryo-EM structure of a fungal mitochondrial calcium uniporter. Nature 559(7715):570–574. Scholar
  50. 50.
    Teo ACK, Lee SC, Pollock NL, Stroud Z, Hall S, Thakker A, Pitt AR, Dafforn TR, Spickett CM, Roper DI (2019) Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein. Sci Rep 9(1):1813. Scholar
  51. 51.
    Parmar M, Rawson S, Scarff CA, Goldman A, Dafforn TR, Muench SP, Postis VLG (2018) Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure. Biochim Biophys Acta 1860(2):378–383. Scholar
  52. 52.
    Sun C, Benlekbir S, Venkatakrishnan P, Wang Y, Hong S, Hosler J, Tajkhorshid E, Rubinstein JL, Gennis RB (2018) Structure of the alternative complex III in a supercomplex with cytochrome oxidase. Nature 557(7703):123–126. Scholar
  53. 53.
    Qiu W, Fu Z, Xu GG, Grassucci RA, Zhang Y, Frank J, Hendrickson WA, Guo Y (2018) Structure and activity of lipid bilayer within a membrane-protein transporter. Proc Natl Acad Sci U S A 115(51):12985–12990. Scholar
  54. 54.
    Swainsbury DJK, Scheidelaar S, Foster N, van Grondelle R, Killian JA, Jones MR (2017) The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes. Biochim Biophys Acta 1859(10):2133–2143. Scholar
  55. 55.
    Dominguez Pardo JJ, Dorr JM, Iyer A, Cox RC, Scheidelaar S, Koorengevel MC, Subramaniam V, Killian JA (2017) Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer. Eur Biophys J 46(1):91–101. Scholar
  56. 56.
    Oluwole AO, Danielczak B, Meister A, Babalola JO, Vargas C, Keller S (2017) Solubilization of membrane proteins into functional lipid‐bilayer nanodiscs using a diisobutylene/maleic acid copolymer. Angew Chem Int Ed Engl. 56(7):1919–1924. Scholar
  57. 57.
    Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2(8):853–856. Scholar
  58. 58.
    Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126(11):3477–3487. Scholar
  59. 59.
    Bayburt TH, Sligar SG (2010) Membrane protein assembly into Nanodiscs. FEBS Lett 584(9):1721–1727. Scholar
  60. 60.
    Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG (2009) Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231. Scholar
  61. 61.
    Bayburt TH, Sligar SG (2003) Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12(11):2476–2481CrossRefGoogle Scholar
  62. 62.
    Schuler MA, Denisov IG, Sligar SG (2013) Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 974:415–433. Scholar
  63. 63.
    Pedemonte N, Galietta LJ (2014) Structure and function of TMEM16 proteins (anoctamins). Physiol Rev 94(2):419–459. Scholar
  64. 64.
    Brunner JD, Lim NK, Schenck S, Duerst A, Dutzler R (2014) X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature 516(7530):207–212. Scholar
  65. 65.
    Paulino C, Kalienkova V, Lam AKM, Neldner Y, Dutzler R (2017) Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM. Nature 552(7685):421–425. Scholar
  66. 66.
    Paulino C, Neldner Y, Lam AK, Kalienkova V, Brunner JD, Schenck S, Dutzler R (2017) Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A. Elife 6:e26232. Scholar
  67. 67.
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1):36–51. Scholar
  68. 68.
    Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, Stagg S, Potter CS, Carragher B (2005) Automated molecular microscopy: the new Leginon system. J Struct Biol 151(1):41–60. Scholar
  69. 69.
    Biyani N, Righetto RD, McLeod R, Caujolle-Bert D, Castano-Diez D, Goldie KN, Stahlberg H (2017) Focus: The interface between data collection and data processing in cryo-EM. J Struct Biol 198(2):124–133. Scholar
  70. 70.
    Scheres SHW (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180(3):519–530. Scholar
  71. 71.
    Grant T, Rohou A, Grigorieff N (2018) cisTEM, user-friendly software for single-particle image processing. Elife 7:e35383. Scholar
  72. 72.
    Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296. Scholar
  73. 73.
    Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157(1):38–46. Scholar
  74. 74.
    Moriya T, Saur M, Stabrin M, Merino F, Voicu H, Huang Z, Penczek PA, Raunser S, Gatsogiannis C (2017) High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE. J Vis Exp 123:55448. Scholar
  75. 75.
    Hahn A, Vonck J, Mills DJ, Meier T, Kühlbrandt W (2018) Structure, mechanism, and regulation of the chloroplast ATP synthase. Science 360(6389):eaat4318. Scholar
  76. 76.
    Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135(5):1919–1925. Scholar
  77. 77.
    Nasr ML, Baptista D, Strauss M, Sun ZJ, Grigoriu S, Huser S, Pluckthun A, Hagn F, Walz T, Hogle JM, Wagner G (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14(1):49–52. Scholar
  78. 78.
    Denisov IG, Baas BJ, Grinkova YV, Sligar SG (2007) Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation. J Biol Chem 282(10):7066–7076. Scholar
  79. 79.
    Grinkova YV, Denisov IG, Sligar SG (2010) Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng Des Sel 23(11):843–848. Scholar
  80. 80.
    Shih AY, Denisov IG, Phillips JC, Sligar SG, Schulten K (2005) Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins. Biophys J 88(1):548–556. Scholar
  81. 81.
    Efremov RG, Gatsogiannis C, Raunser S (2017) Lipid nanodiscs as a tool for high-resolution structure determination of membrane proteins by single-particle cryo-EM. Methods Enzymol 594:1–30. Scholar
  82. 82.
    Denisov IG, McLean MA, Shaw AW, Grinkova YV, Sligar SG (2005) Thermotropic phase transition in soluble nanoscale lipid bilayers. J Phys Chem B 109(32):15580–15588. Scholar
  83. 83.
    Kawai T, Caaveiro JMM, Abe R, Katagiri T, Tsumoto K (2011) Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer. FEBS Lett 585(22):3533–3537. Scholar
  84. 84.
    Nasr ML, Wagner G (2018) Covalently circularized nanodiscs; challenges and applications. Curr Opin Struct Biol 51:129–134. Scholar
  85. 85.
    Johansen NT, Tidemand FG, Nguyen TTTN, Rand KD, Pedersen MC, Arleth L (2019) Circularized and solubility-enhanced MSPs facilitate simple and high-yield production of stable nanodiscs for studies of membrane proteins in solution. Febs Journal 286(9):1734–1751.
  86. 86.
    Hagn F, Nasr ML, Wagner G (2018) Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13(1):79–98. Scholar
  87. 87.
    Naydenova K, Russo CJ (2017) Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. Nat Commun 8(1):629. Scholar
  88. 88.
    Tan YZ, Baldwin PR, Davis JH, Williamson JR, Potter CS, Carragher B, Lyumkis D (2017) Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat Methods 14(8):793–796. Scholar
  89. 89.
    Baldwin PR, Lyumkis D (2019) Non-uniformity of projection distributions attenuates resolution in cryo-EM. bioRxiv:635938.
  90. 90.
    Glaeser RM, Han B-G (2017) Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys Rep 3(1):1–7. Scholar
  91. 91.
    Taylor KA, Glaeser RM (2008) Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. J Struct Biol 163(3):214–223. Scholar
  92. 92.
    Noble AJ, Wei H, Dandey VP, Zhang Z, Tan YZ, Potter CS, Carragher B (2018) Reducing effects of particle adsorption to the air–water interface in cryo-EM. Nat Methods 15(10):793–795. Scholar
  93. 93.
    Efremov RG, Leitner A, Aebersold R, Raunser S (2014) Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517:39–43. Scholar
  94. 94.
    Johnson ZL, Chen J (2017) Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 168(6):1075–1085. Scholar
  95. 95.
    Zhang Z, Chen J (2016) Atomic structure of the cystic fibrosis transmembrane conductance regulator. Cell 167(6):1586–1597. Scholar
  96. 96.
    Russo CJ, Passmore LA (2014) Ultrastable gold substrates for electron cryomicroscopy. Science 346(6215):1377–1380. Scholar
  97. 97.
    Russo CJ, Passmore LA (2016) Ultrastable gold substrates: properties of a support for high-resolution electron cryomicroscopy of biological specimens. J Struct Biol 193(1):33–44. Scholar
  98. 98.
    Meyerson JR, Rao P, Kumar J, Chittori S, Banerjee S, Pierson J, Mayer ML, Subramaniam S (2014) Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Sci Rep 4:7084. Scholar
  99. 99.
    Nguyen THD, Galej WP, Bai X-c, Savva CG, Newman AJ, Scheres SHW, Nagai K (2015) The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523:47–52. Scholar
  100. 100.
    Llaguno MC, Xu H, Shi L, Huang N, Zhang H, Liu Q, Jiang Q-X (2014) Chemically functionalized carbon films for single molecule imaging. J Struct Biol 185(3):405–417. Scholar
  101. 101.
    Palovcak E, Wang F, Zheng SQ, Yu Z, Li S, Betegon M, Bulkley D, Agard DA, Cheng Y (2018) A simple and robust procedure for preparing graphene-oxide cryo-EM grids. J Struct Biol 204(1):80–84. Scholar
  102. 102.
    D’Imprima E, Floris D, Joppe M, Sánchez R, Grininger M, Kühlbrandt W (2019) Protein denaturation at the air-water interface and how to prevent it. Elife 8:e42747. Scholar
  103. 103.
    Liu N, Zhang J, Chen Y, Liu C, Zhang X, Xu K, Wen J, Luo Z, Chen S, Gao P, Jia K, Liu Z, Peng H, Wang H-W (2019) Bioactive functionalized monolayer graphene for high-resolution cryo-electron microscopy. J Am Chem Soc 141(9):4016–4025. Scholar
  104. 104.
    Naydenova K, Peet MJ, Russo CJ (2019) Multifunctional graphene supports for electron cryomicroscopy. Proc Natl Acad Sci U S A:11718–11724.
  105. 105.
    Yu G, Li K, Huang P, Jiang X, Jiang W (2016) Antibody-based affinity cryoelectron microscopy at 2.6-Å resolution. Structure 24(11):1984–1990. Scholar
  106. 106.
    Yu G, Li K, Jiang W (2016) Antibody-based affinity cryo-EM grid. Methods 100:16–24. Scholar
  107. 107.
    Glaeser RM (2018) Proteins, interfaces and cryo-EM grids. Curr Opin Colloid Interface Sci 34:1–8. Scholar
  108. 108.
    Han BG, Watson Z, Cate JHD, Glaeser RM (2017) Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality. J Struct Biol 200(3):307–313. Scholar
  109. 109.
    Wang F, Liu Y, Yu Z, Li S, Cheng Y, Agard DA (2019) General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM. bioRxiv:657411.
  110. 110.
    Basu K, Green EM, Cheng Y, Craik CS (2019) Why recombinant antibodies—benefits and applications. Curr Opin Biotechnol 60:153–158. Scholar
  111. 111.
    Masiulis S, Desai R, Uchański T, Serna Martin I, Laverty D, Karia D, Malinauskas T, Zivanov J, Pardon E, Kotecha A, Steyaert J, Miller KW, Aricescu AR (2019) GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature 565(7740):454–459. Scholar
  112. 112.
    Uchański T, Masiulis S, Fischer B, Kalichuk V, Wohlkönig A, Zögg T, et al. (2019) Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. bioRxiv 343, 812230.
  113. 113.
    Dandey VP, Wei H, Zhang Z, Tan YZ, Acharya P, Eng ET, Rice WJ, Kahn PA, Potter CS, Carragher B (2018) Spotiton: new features and applications. J Struct Biol 202(2):161–169. Scholar
  114. 114.
    Jain T, Sheehan P, Crum J, Carragher B, Potter CS (2012) Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J Struct Biol 179(1):68–75. Scholar
  115. 115.
    Wei H, Dandey VP, Zhang Z, Raczkowski A, Rice WJ, Carragher B, Potter CS (2018) Optimizing “self-wicking” nanowire grids. J Struct Biol 202(2):170–174. Scholar
  116. 116.
    Razinkov I, Dandey V, Wei H, Zhang Z, Melnekoff D, Rice WJ, Wigge C, Potter CS, Carragher B (2016) A new method for vitrifying samples for cryoEM. J Struct Biol 195(2):190–198. Scholar
  117. 117.
    Ravelli RBG, Nijpels FJT, Henderikx RJM, Weissenberger G, Thewessem S, Gijsbers A, Beulen BWAMM, López-Iglesias C, Peters PJ (2019) Automated cryo-EM sample preparation by pin-printing and jet vitrification. bioRxiv:651208.
  118. 118.
    Arnold SA, Albiez S, Bieri A, Syntychaki A, Adaixo R, McLeod RA, Goldie KN, Stahlberg H, Braun T (2017) Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts. J Struct Biol 197(3):220–226. Scholar
  119. 119.
    Snijder J, Borst AJ, Dosey A, Walls AC, Burrell A, Reddy VS, Kollman JM, Veesler D (2017) Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids. J Struct Biol 198(1):38–42. Scholar
  120. 120.
    Kim LY, Rice WJ, Eng ET, Kopylov M, Cheng A, Raczkowski AM, Jordan KD, Bobe D, Potter CS, Carragher B (2018) Benchmarking cryo-EM single particle analysis workflow. Front Mol Biosci 5:50. Scholar
  121. 121.
    Downing KH, Glaeser RM (2018) Estimating the effect of finite depth of field in single-particle cryo-EM. Ultmi 184(Pt A):94–99. Scholar
  122. 122.
    Rice WJ, Cheng A, Noble AJ, Eng ET, Kim LY, Carragher B, Potter CS (2018) Routine determination of ice thickness for cryo-EM grids. J Struct Biol 204(1):38–44. Scholar
  123. 123.
    Penczek PA (2010) Chapter 1: Fundamentals of three-dimensional reconstruction from projections. In: Jensen GJ (ed) Methods in enzymology, vol 482. Academic Press, New York, pp 1–33. Scholar
  124. 124.
    Radermacher M (1988) Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J Electron Microsc Tech 9(4):359–394. Scholar
  125. 125.
    Radermacher M, Wagenknecht T, Verschoor A, Frank J (1987) Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J Microsc 146(2):113–136. Scholar
  126. 126.
    Leschziner AE, Nogales E (2006) The orthogonal tilt reconstruction method: an approach to generating single-class volumes with no missing cone for ab initio reconstruction of asymmetric particles. J Struct Biol 153(3):284–299. Scholar
  127. 127.
    Asarnow D, Palovcak E, Gao Y, Julius D, Cheng Y (2017) Ion channel in lipid nanodisc by single particle cryo-EM – pushing the technology limit. Microsc Microanal 23(S1):822–823. Scholar
  128. 128.
    von Loeffelholz O, Natchiar SK, Djabeur N, Myasnikov AG, Kratzat H, Ménétret J-F, Hazemann I, Klaholz BP (2017) Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes. Curr Opin Struct Biol 46:140–148. Scholar
  129. 129.
    Scheres SHW (2016) Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol 579:125–157. Scholar
  130. 130.
    Bai XC, Rajendra E, Yang G, Shi Y, Scheres SHW (2015) Sampling the conformational space of the catalytic subunit of human γ-secretase. Elife 4:e11182. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Valeria Kalienkova
    • 1
    • 2
  • Carolina Alvadia
    • 2
  • Vanessa Clerico Mosina
    • 1
  • Cristina Paulino
    • 1
    Email author
  1. 1.Department of Structural Biology at the Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenGroningenThe Netherlands
  2. 2.Department of BiochemistryUniversity of ZurichZurichSwitzerland

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