Volume 7 Issue 3
Jun.  2021
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Article Contents
Nan Liu, Liming Zheng, Jie Xu, Jia Wang, Cuixia Hu, Jun Lan, Xing Zhang, Jincan Zhang, Kui Xu, Hang Cheng, Zi Yang, Xin Gao, Xinquan Wang, Hailin Peng, Yanan Chen, Hong-Wei Wang. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM[J]. Biophysics Reports, 2021, 7(3): 227-238. doi: 10.52601/bpr.2021.210007
Citation: Nan Liu, Liming Zheng, Jie Xu, Jia Wang, Cuixia Hu, Jun Lan, Xing Zhang, Jincan Zhang, Kui Xu, Hang Cheng, Zi Yang, Xin Gao, Xinquan Wang, Hailin Peng, Yanan Chen, Hong-Wei Wang. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM[J]. Biophysics Reports, 2021, 7(3): 227-238. doi: 10.52601/bpr.2021.210007

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM

doi: 10.52601/bpr.2021.210007
Funds:  We thank Dr. Ning Gao, Dr. Xueming Li for kindly providing ribosome and 20S proteasome samples. We are grateful to Dr. Jianlin Lei, Dr. Lingpeng Cheng, Dr. Tao Yang, Dr. Xiaomin Li, Dr. Fan Yang, Danyang Li, Xiaofeng Hu, Jie Wen, Yakun Wang, and Anbao Jia at the Cryo-EM and High-Performance Computation platforms of Tsinghua University Branch of the National Protein Science Facility, for the technical support in cryo-EM data collection and analysis. This work is financially supported by the Ministry of Science and Technology of China (2016YFA0501100) and National Natural Science Foundation of China (31825009) to H-W Wang, and the National Natural Science Foundation of China (21525310) and the National Basic Research Program of China (2014CB932500 and 2016YFA0200101) to H Peng.
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  • Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.
  • † Nan Liu, Liming Zheng, Jie Xu and Jia Wang contributed equally to this work
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  • [1]
    Armstrong M, Han BG, Gomez S, Turner J, Fletcher DA, Glaeser RM (2020) Microscale fluid behavior during cryo-EM sample blotting. Biophys J 118(3): 708−719 doi: 10.1016/j.bpj.2019.12.017
    [2]
    Bai R, Wan R, Yan C, Jia Q, Lei J, Shi Y (2020) Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2. Science 371(6525): eabe8863. https://doi.org/10.1126/science.abe8863
    [3]
    Balandin AA, Ghosh S, Bao WZ, Calizo I, Teweldebrhan D, Miao F, Lau CN (2008) Superior thermal conductivity of single-layer graphene. Nano Letters 8(3): 902−907 doi: 10.1021/nl0731872
    [4]
    Benjamin CJ, Wright KJ, Bolton SC, Hyun SH, Krynski K, Grover M, Yu GM, Guo F, Kinzer-Ursem TL, Jiang W, Thompson DH (2016) Selective capture of histidine-tagged proteins from cell lysates using TEM grids modified with NTA-graphene oxide. Sci Rep 6: 32500. https://doi.org/10.1038/srep32500
    [5]
    Brink J, Sherman MB, Berriman J, Chiu W (1998) Evaluation of charging on macromolecules in electron cryomicroscopy. Ultramicroscopy 72(1-2): 41−52 doi: 10.1016/S0304-3991(97)00126-5
    [6]
    Buchsteiner A, Lerf A, Pieper J (2006) Water dynamics in graphite oxide investigated with neutron scattering. J Phys Chem B 110(45): 22328−22338 doi: 10.1021/jp0641132
    [7]
    Chen JH, Jang C, Adam S, Fuhrer MS, Williams ED, Ishigami M (2008) Charged-impurity scattering in graphene. Nat Phys 4(5): 377−381 doi: 10.1038/nphys935
    [8]
    Cheng Y (2015) Single-particle cryo-EM at crystallographic resolution. Cell 161(3): 450−457 doi: 10.1016/j.cell.2015.03.049
    [9]
    Cheng YF (2018) Single-particle cryo-EM—How did it get here and where will it go. Science 361(6405): 876−880 doi: 10.1126/science.aat4346
    [10]
    Choppin G, Liljenzin J-O, Rydberg J, Ekberg C (2013) Absorption of nuclear radiation. In: Radiochemistry and Nuclear Chemistry. pp 163-208
    [11]
    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. https://doi.org/10.7554/eLife.42747
    [12]
    Danev R, Buijsse B, Khoshouei M, Plitzko JM, Baumeister W (2014) Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc Natl Acad Sci USA 111(44): 15635−15640 doi: 10.1073/pnas.1418377111
    [13]
    Dubochet J, Lepault J, Freeman R, Berriman JA, Homo JC (1982) Electron-microscopy of frozen water and aqueous-solutions. J Microsc 128(Dec): 219−237
    [14]
    Egerton RF, Li P, Malac M (2004) Radiation damage in the TEM and SEM. Micron 35(6): 399−409 doi: 10.1016/j.micron.2004.02.003
    [15]
    Fan X, Zhao L, Liu C, Zhang JC, Fan K, Yan X, Peng HL, Lei J, Wang HW (2017) Near-atomic resolution structure determination in over-focus with Volta phase plate by Cs-corrected cryo-EM. Structure 25(10): 1623−1630 doi: 10.1016/j.str.2017.08.008
    [16]
    Geim AK, Novoselov KS (2007) The rise of graphene. Nat Mater 6(3): 183−191 doi: 10.1038/nmat1849
    [17]
    Glaeser RM (2016) Specimen behavior in the electron beam. Methods Enzymol 579: 19−50
    [18]
    Glaeser RM (2018) Proteins, interfaces, and cryo-EM grids. Curr Opin Colloid Interface Sci 34: 1−8 doi: 10.1016/j.cocis.2017.12.009
    [19]
    Glaeser RM, Han BG (2017) Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys Rep 3(1): 1−7
    [20]
    Grassucci RA, Taylor DJ, Frank J (2007) Preparation of macromolecular complexes for cryo-electron microscopy. Nat Protoc 2(12): 3239−3246 doi: 10.1038/nprot.2007.452
    [21]
    Han Y, Fan X, Wang H, Zhao F, Tully CG, Kong J, Yao N, Yan N (2020) High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy. Proc Natl Acad Sci USA 117(2): 1009−1014 doi: 10.1073/pnas.1919114117
    [22]
    Hettler S, Kano E, Dries M, Gerthsen D, Pfaffmann L, Bruns M, Beleggia M, Malac M (2018) Charging of carbon thin films in scanning and phase-plate transmission electron microscopy. Ultramicroscopy 184: 252−266 doi: 10.1016/j.ultramic.2017.09.009
    [23]
    Jiang N, Spence JCH (2009) Radiation damage in zircon by high-energy electron beams. J Appl Phys 105(12): 123517. https://doi.org/10.1063/1.3151704
    [24]
    Jung I, Dikin DA, Piner RD, Ruoff RS (2008) Tunable electrical conductivity of individual graphene oxide sheets reduced at "low" temperatures. Nano Lett 8(12): 4283−4287 doi: 10.1021/nl8019938
    [25]
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1): 71−76 doi: 10.1006/jsbi.1996.0013
    [26]
    Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, Zhang Q, Shi X, Wang Q, Zhang L, Wang X (2020) Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581(7807): 215−220 doi: 10.1038/s41586-020-2180-5
    [27]
    Lee C, Wei XD, Kysar JW, Hone J (2008) Measurement of the elastic properties and intrinsic strength of monolayer graphene. Science 321(5887): 385−388 doi: 10.1126/science.1157996
    [28]
    Lei J, Frank J (2005) Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J Struct Biol 150(1): 69−80 doi: 10.1016/j.jsb.2005.01.002
    [29]
    Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB, Gubbens S, Agard DA, Cheng Y (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10(6): 584−590 doi: 10.1038/nmeth.2472
    [30]
    Lian B, De Luca S, You Y, Alwarappan S, Yoshimura M, Sahajwalla V, Smith SC, Leslie G, Joshi RK (2018) Extraordinary water adsorption characteristics of graphene oxide. Chem Sci 9(22): 5106−5111 doi: 10.1039/C8SC00545A
    [31]
    Lin L, Sun LZ, Zhang JC, Sun JY, Koh AL, Peng HL, Liu ZF (2016) Rapid growth of large single-crystalline graphene via second passivation and multistage carbon supply. Adv Mater 28(23): 4671−4677 doi: 10.1002/adma.201600403
    [32]
    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 HW (2019) Bioactive functionalized monolayer graphene for high-resolution cryo-electron microscopy. J Am Chem Soc 141(9): 4016−4025 doi: 10.1021/jacs.8b13038
    [33]
    Ma CY, Wu S, Li NN, Chen Y, Yan KG, Li ZF, Zheng LQ, Lei JL, Woolford JL, Gao N (2017) Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1. Nat Struct Molr Biol 24(3): 214−220 doi: 10.1038/nsmb.3364
    [34]
    Marcano DC, Kosynkin DV, Berlin JM, Sinitskii A, Sun Z, Slesarev A, Alemany LB, Lu W, Tour JM (2010) Improved synthesis of graphene oxide. ACS Nano 4(8): 4806−4814 doi: 10.1021/nn1006368
    [35]
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152(1): 36−51 doi: 10.1016/j.jsb.2005.07.007
    [36]
    Moon IK, Lee J, Ruoff RS, Lee H (2010) Reduced graphene oxide by chemical graphitization. Nat Commun 1: 73. https://doi.org/10.1038/ncomms1067
    [37]
    Nakane T, Kotecha A, Sente A, McMullan G, Masiulis S, Brown PMGE, Grigoras IT, Malinauskaite L, Malinauskas T, Miehling J, Yu L, Karia D, Pechnikova EV, de Jong E, Keizer J, Bischoff M, McCormack J, Tiemeijer P, Hardwick SW, Chirgadze DY, Murshudov G, Aricescu AR, Scheres SHW (2020) Single-particle cryo-EM at atomic resolution. Nature 587(7832): 152−156 doi: 10.1038/s41586-020-2829-0
    [38]
    Naydenova K, Peet MJ, Russo CJ (2019) Multifunctional graphene supports for electron cryomicroscopy. Proc Natl Acad Sci USA 116(24): 11718−11724
    [39]
    Naydenova K, Russo CJ (2017) Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. Nat Commun 8(1): 629. https://doi.org/10.1038/s41467-017-00782-3
    [40]
    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 doi: 10.1038/s41592-018-0139-3
    [41]
    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 doi: 10.1016/j.jsb.2018.07.007
    [42]
    Pantelic RS, Meyer JC, Kaiser U, Baumeister W, Plitzko JM (2010) Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples. J Struct Biol 170(1): 152−156 doi: 10.1016/j.jsb.2009.12.020
    [43]
    Peet MJ, Henderson R, Russo CJ (2019) The energy dependence of contrast and damage in electron cryomicroscopy of biological molecules. Ultramicroscopy 203: 125−131 doi: 10.1016/j.ultramic.2019.02.007
    [44]
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera — A visualization system for exploratory research and analysis. J Comput Chem 25(13): 1605−1612 doi: 10.1002/jcc.20084
    [45]
    Qiu TF, Luo B, Liang MH, Ning J, Wang B, Li XL, Zhi LJ (2015) Hydrogen reduced graphene oxide/metal grid hybrid film: towards high performance transparent conductive electrode for flexible electrochromic devices. Carbon 81: 232−238 doi: 10.1016/j.carbon.2014.09.054
    [46]
    Regan W, Alem N, Aleman B, Geng BS, Girit C, Maserati L, Wang F, Crommie M, Zettl A (2010) A direct transfer of layer-area graphene. Appl Phys Lett 96(11): 113102. https://doi.org/10.1063/1.3337091
    [47]
    Rohou A, Grigorieff N (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct Biol 192(2): 216−221 doi: 10.1016/j.jsb.2015.08.008
    [48]
    Russo CJ, Passmore LA (2014a) Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas. Nat Methods 11(6): 649−652 doi: 10.1038/nmeth.2931
    [49]
    Russo CJ, Passmore LA (2014b) Ultrastable gold substrates for electron cryomicroscopy. Science 346(6215): 1377−1380 doi: 10.1126/science.1259530
    [50]
    Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180(3): 519−530 doi: 10.1016/j.jsb.2012.09.006
    [51]
    Scheres SH (2016) Processing of structurally heterogeneous cryo-EM Data in RELION. Methods Enzymol 579: 125−157
    [52]
    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 doi: 10.1038/nmeth.4347
    [53]
    Wang F, Yu Z, Betegon M, Campbell MG, Aksel T, Zhao J, Li S, Douglas SM, Cheng Y, Agard DA (2020) Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy. J Struct Biol 209(2): 107437. https://doi.org/10.1016/j.jsb.2019.107437
    [54]
    Wang YL, Chen YA, Lacey SD, Xu LS, Xie H, Li T, Danner VA, Hu LB (2018) Reduced graphene oxide film with record-high conductivity and mobility. Mater Today 21(2): 186−192 doi: 10.1016/j.mattod.2017.10.008
    [55]
    Wilson NR, Pandey PA, Beanland R, Young RJ, Kinloch IA, Gong L, Liu Z, Suenaga K, Rourke JP, York SJ, Sloan J (2009) Graphene oxide: structural analysis and application as a highly transparent support for electron microscopy. Acs Nano 3(9): 2547−2556 doi: 10.1021/nn900694t
    [56]
    Wu S, Armache JP, Cheng Y (2016) Single-particle cryo-EM data acquisition by using direct electron detection camera. Microscopy (Oxf) 65(1): 35−41
    [57]
    Yip KM, Fischer N, Paknia E, Chari A, Stark H (2020) Breaking the next Cryo-EM resolution barrier — Atomic resolution determination of proteins! BioRxiv: 10.1101/2020.05.21.106740
    [58]
    You S, Sundqvist B, Talyzin AV (2013) Enormous lattice expansion of hummers graphite oxide in alcohols at low temperatures. ACS Nano 7(2): 1395−1399 doi: 10.1021/nn3051105
    [59]
    Zhang J, Lin L, Sun L, Huang Y, Koh AL, Dang W, Yin J, Wang M, Tan C, Li T, Tan Z, Liu Z, Peng H (2017) Clean transfer of large graphene single crystals for high-intactness suspended membranes and liquid cells. Adv Mater 29(26): 1700639. https://doi.org/10.1002/adma.201700639
    [60]
    Zhang XY, Huang Y, Wang Y, Ma YF, Liu ZF, Chen YS (2009) Synthesis and characterization of a graphene-C-60 hybrid material. Carbon 47(1): 334−337 doi: 10.1016/j.carbon.2008.10.018
    [61]
    Zheng L, Chen Y, Li N, Zhang J, Liu N, Liu J, Dang W, Deng B, Li Y, Gao X, Tan C, Yang Z, Xu S, Wang M, Yang H, Sun L, Cui Y, Wei X, Gao P, Wang HW, Peng H (2020) Robust ultraclean atomically thin membranes for atomic-resolution electron microscopy. Nat Commun 11(1): 541. https://doi.org/10.1038/s41467-020-14359-0
    [62]
    Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14(4): 331−332 doi: 10.1038/nmeth.4193
    [63]
    Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579(7798): 270−273 doi: 10.1038/s41586-020-2012-7
    [64]
    Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJ, Lindahl E, Scheres SH (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7: e42166. https://doi.org/10.7554/eLife.42166
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