Theoretical framework and experimental solution for the air−water interface adsorption problem in cryoEM

Joon S. Kang; Xueting Zhou; Yun-Tao Liu; Kaituo Wang; Z. Hong Zhou

doi:10.52601/bpr.2023.230008

Volume 9 Issue 4

Aug. 2023

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Joon S. Kang, Xueting Zhou, Yun-Tao Liu, Kaituo Wang, Z. Hong Zhou. Theoretical framework and experimental solution for the air−water interface adsorption problem in cryoEM. Biophysics Reports, 2023, 9(4): 215-229. doi: 10.52601/bpr.2023.230008

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Joon S. Kang, Xueting Zhou, Yun-Tao Liu, Kaituo Wang, Z. Hong Zhou. Theoretical framework and experimental solution for the air−water interface adsorption problem in cryoEM. Biophysics Reports, 2023, 9(4): 215-229. doi: 10.52601/bpr.2023.230008

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Theoretical framework and experimental solution for the air−water interface adsorption problem in cryoEM

doi: 10.52601/bpr.2023.230008

Joon S. Kang^{1, 2, J},
Xueting Zhou^{1, J},
Yun-Tao Liu^{1, 3},
Kaituo Wang^{1, 3},
Z. Hong Zhou^{1, 2, 3
,
,}

1.
Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA
2.
Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA
3.
California NanoSystems Institute, UCLA, Los Angeles, CA 90095, USA

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Author Bio:
Joon S. Kang and
Corresponding author: Hong.Zhou@UCLA.edu
Received Date: 07 October 2023
Accepted Date: 31 October 2023

Available Online: 20 December 2023

Publish Date: 31 August 2023

Abstract

Abstract

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously “disappear” on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air–water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM.
- CryoEM,
- CryoET,
- Sample preparation,
- Air–water interface adsorption,
- Surfactant,
- Surface energy

Joon S. Kang, Xueting Zhou, Yun-Tao Liu, Kaituo Wang and Z. Hong Zhou declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
Joon S. Kang and Xueting Zhou contributed equally to this work.

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References(54)

References

Bai XC, Fernandez IS, McMullan G, Scheres SH (2013) Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. Elife 2: e0046. https://doi.org/10.7554/eLife.00461

Chamberlain AK, Handel TM, Marqusee S (1996) Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH. Nat Struct Biol 3(9): 782−787 doi: 10.1038/nsb0996-782

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 https://doi.org/10.1016/j.yjsbx.2019.100005

Chen S, Li J, Vinothkumar KR, Henderson R (2022) Interaction of human erythrocyte catalase with air–water interface in cryoEM. Microscopy (Oxf) 71(Supplement_1): i51−i59

Chu CH, Sarangadharan I, Regmi A, Chen YW, Hsu CP, Chang WH, Lee GY, Chyi JI, Chen CC, Shiesh SC, Lee GB, Wang YL (2017) Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum. Sci Rep 7(1): 5256. https://doi.org/10.1038/s41598-017-05426-6

Cieplak M, Allan DB, Leheny RL, Reich DH (2014) Proteins at air-water interfaces: a coarse-grained model. Langmuir 30(43): 12888−12896 doi: 10.1021/la502465m

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. https://doi.org/10.7554/eLife.42747

Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys. 21:129 − 228

Fan H, Wang B, Zhang Y, Zhu Y, Song B, Xu H, Zhai Y, Qiao M, Sun F (2021) A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI. Nat Commun 12(1): 7257. https://doi.org/10.1038/s41467-021-27596-8

Glaeser RM (2018) Proteins, interfaces, and cryo-EM grids. Curr Opin Colloid Interface Sci 34:1−8

Glaeser RM, Han B-G (2017) Opinion: hazards faced by macromolecules when confined to thin aqueous films. Biophys Rep 3(1): 1−7

Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE (2018) UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci 27(1): 14−25 doi: 10.1002/pro.3235

Graham DE, Phillips MC (1979) Proteins at liquid interfaces: I. Kinetics of adsorption and surface denaturation. J Colloid Interface Sci 70(3): 403−414

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

Hauner IM, Deblais A, Beattie JK, Kellay H, Bonn D (2017) The dynamic surface tension of water. J Phys Chem Lett 8(7): 1599−1603 doi: 10.1021/acs.jpclett.7b00267

Hiemenz P, Rajagopalan R (Eds) (1997) Principles of colloid and surface chemistry, revised and expanded. CRC Press. https://doi.org/10.1201/9781315274287

Hoffmann PC, Kreysing JP, Khusainov I, Tuijtel MW, Welsch S, Beck M (2022) Structures of the eukaryotic ribosome and its translational states in situ. Nat Commun 13(1): 7435. https://doi.org/10.1038/s41467-022-34997-w

Hughes TET, Lodowski DT, Huynh KW, Yazici A, Del Rosario J, Kapoor A, Basak S, Samanta A, Han X, Chakrapani S, Zhou ZH, Filizola M, Rohacs T, Han S, Moiseenkova-Bell VY (2018) Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat Struct Mol Biol 25(1): 53−60 doi: 10.1038/s41594-017-0009-1

Inácio ÂS, Mesquita KA, Baptista M, Ramalho-Santos J, Vaz WLC, Vieira OV (2011) In vitro surfactant structure-toxicity relationships: implications for surfactant use in sexually transmitted infection prophylaxis and contraception. PLoS One 6(5): e19850−e19850 doi: 10.1371/journal.pone.0019850

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 doi: 10.1016/j.jsb.2012.04.020

Johnson ZL, Chen J (2017) Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 168(6): 1075 − 1085 e1079

Kirby BJ (2010) Micro- and nanoscale fluid mechanics: transport in microfluidic devices. Cambridge University Press. https://doi.org/10.1017/CBO9780511760723

Koepf E, Schroeder R, Brezesinski G, Friess W (2017) The film tells the story: physical-chemical characteristics of IgG at the liquid-air interface. Eur J Pharm Biopharm 119: 396−407 doi: 10.1016/j.ejpb.2017.07.006

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

Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504(7478): 107−112 doi: 10.1038/nature12822

Liao W-C, Zatz JL (1979) Surfactant solutions as test liquids for measurement of critical surface tension. J Pharm Sci 68(4): 486−488 doi: 10.1002/jps.2600680425

Lyumkis D (2019) Challenges and opportunities in cryo-EM single-particle analysis. J Biol Chem 294(13): 5181−5197 doi: 10.1074/jbc.REV118.005602

MacRitchie F (1985) Desorption of proteins from the air/water interface. J Colloid Interface Sci 105(1): 119−123 doi: 10.1016/0021-9797(85)90353-4

Maity H, Maity M, Krishna MMG, Mayne L, Englander SW (2005) Protein folding: the stepwise assembly of foldon units. Proc Natl Acad Sci USA 102(13): 4741−4746 doi: 10.1073/pnas.0501043102

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

Mondal S, Phukan M, Ghatak A (2015) Estimation of solid-liquid interfacial tension using curved surface of a soft solid. Proc Natl Acad Sci USA 112: 12563−12568

Narsimhan G, Uraizee F (1992) Kinetics of adsorption of globular proteins at an air–water interface. Biotechnol Prog. 8(3): 187−196 doi: 10.1021/bp00015a003

Naydenova K, Russo CJ (2017) Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. Nature Commun 8(1): 629. https://doi.org/10.1038/s41467-017-00782-3

Noble AJ, Dandey VP, Wei H, Brasch J, Chase J, Acharya P, Tan YZ, Zhang Z, Kim LY, Scapin G, Rapp M, Eng ET, Rice WJ, Cheng A, Negro CJ, Shapiro L, Kwong PD, Jeruzalmi D, des Georges A, Potter CS, Carragher B (2018a) Routine single particle CryoEM sample and grid characterization by tomography. Elife 29(7): e34257. https://doi.org/10.7554/eLife.34257

Noble AJ, Wei H, Dandey VP, Zhang Z, Tan YZ, Potter CS, Carragher B (2018b) 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

O'Reilly FJ, Xue L, Graziadei A, Sinn L, Lenz S, Tegunov D, Blotz C, Singh N, Hagen WJH, Cramer P, Stulke J, Mahamid J, Rappsilber J (2020) In-cell architecture of an actively transcribing-translating expressome. Science 369(6503): 554−557 doi: 10.1126/science.abb3758

Pantelic RS, Suk JW, Magnuson CW, Meyer JC, Wachsmuth P, Kaiser U, Ruoff RS, Stahlberg H (2011) Graphene: substrate preparation and introduction. J Struct Biol 174(1): 234−238 doi: 10.1016/j.jsb.2010.10.002

Park KH, Berrier C, Lebaupain F, Pucci B, Popot JL, Ghazi A, Zito F (2007) Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis. Biochem J 403(1): 183−187 doi: 10.1042/BJ20061473

Roh S-H, Hryc CF, Jeong H-H, Fei X, Jakana J, Lorimer GH, Chiu W (2017) Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM. Proc Natl Acad Sci USA 114(31): 8259−8264 doi: 10.1073/pnas.1704725114

Russo CJ, Passmore LA (2014) Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas. Nat Methods 11(6): 649−652 doi: 10.1038/nmeth.2931

Russo CJ, Passmore LA (2016) Progress towards an optimal specimen support for electron cryomicroscopy. Curr Opin Struct Biol 37: 81−89 doi: 10.1016/j.sbi.2015.12.007

Shuttleworth R (1950) The surface tension of solids. Proc Natl Acad Sci USA 63: 444−457

Stauber T, Weinert S, Jentsch TJ (2012) Cell biology and physiology of CLC chloride channels and transporters. Compr Physiol 2(3): 1701−1744

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 doi: 10.1016/j.jsb.2008.06.004

Tegunov D, Cramer P (2019) Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods 16(11): 1146−1152 doi: 10.1038/s41592-019-0580-y

Tegunov D, Xue L, Dienemann C, Cramer P, Mahamid J (2021) Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells. Nat Methods 18(2): 186 − 193

Temam R (2001) Navier-Stokes Equations: Theory and Numerical Analysis. https://doi.org/10.1090/chel/343

Tribet C, Audebert R, Popot J-L (1996) Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93(26): 15047. https://doi.org/10.1073/pnas.93.26.15047

Vinothkumar KR (2015) Membrane protein structures without crystals, by single particle electron cryomicroscopy. Curr Opin Struct Biol 33: 103−114 doi: 10.1016/j.sbi.2015.07.009

Wang J, Chen L (2003) Domain motions in GroEL upon binding of an oligopeptide. J Mol Biol. 334(3): 489−499 doi: 10.1016/j.jmb.2003.09.074

Wang K, Preisler SS, Zhang L, Cui Y, Missel JW, Gronberg C, Gotfryd K, Lindahl E, Andersson M, Calloe K, Egea PF, Klaerke DA, Pusch M, Pedersen PA, Zhou ZH, Gourdon P (2019) Structure of the human ClC-1 chloride channel. PLoS Biol 17(4): e3000218. https://doi.org/10.1371/journal.pbio.3000218

Wiesbauer J, Prassl R, Nidetzky B (2013) Renewal of the air–water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface–active compounds. Langmuir 29(49): 15240−15250 doi: 10.1021/la4028223

Williams RC, Glaeser RM (1972) Ultrathin carbon support films for electron microscopy. Science 175(4025): 1000−1001 doi: 10.1126/science.175.4025.1000

Zhao Y, Chwastyk M, Cieplak M (2017) Topological transformations in proteins: effects of heating and proximity of an interface. Sci Rep 7(1): 39851. https://doi.org/10.1038/srep39851

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