Hostname: page-component-78c5997874-fbnjt Total loading time: 0 Render date: 2024-11-10T06:08:41.007Z Has data issue: false hasContentIssue false
  • English
  • Français

Minimizing Crinkling of Soft Specimens Using Holey Gold Films on Molybdenum Grids for Cryogenic Electron Microscopy

Published online by Cambridge University Press:  04 June 2021

Xi Jiang Open the ORCID record for Xi Jiang [Opens in a new window] ,
Sunting Xuan ,
Ronald N. Zuckermann ,
Robert M. Glaeser ,
Kenneth H. Downing  and
Nitash P. Balsara
Xi Jiang*
Affiliation:
Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA
Sunting Xuan
Affiliation:
Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA
Ronald N. Zuckermann
Affiliation:
Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA
Robert M. Glaeser
Affiliation:
Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA
Kenneth H. Downing
Affiliation:
Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA
Nitash P. Balsara
Affiliation:
Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA94720, USA
*
*Author for correspondence: Xi Jiang, E-mail: xijiang@lbl.gov
Get access

Abstract

We introduce a novel composite holey gold support that prevents cryo-crinkling and reduces beam-induced motion of soft specimens, building on the previously introduced all-gold support. The composite holey gold support for high-resolution cryogenic electron microscopy of soft crystalline membranes was fabricated in two steps. In the first step, a holey gold film was transferred on top of a molybdenum grid. In the second step, a continuous thin carbon film was transferred onto the holey gold film. This support (Au/Mo grid) was used to image crystalline synthetic polymer membranes. The low thermal expansion of Mo is not only expected to avoid cryo-crinkling of the membrane when the grids are cooled to cryogenic temperatures, but it may also act to reduce whatever crinkling existed even before cooling. The Au/Mo grid exhibits excellent performance with specimens tilted to 45°. This is demonstrated by quantifying beam-induced motion and differences in local defocus values. In addition, images of specimens on the Au/Mo grids that are tilted at 45° show high-resolution information of the crystalline membranes that, after lattice-unbending, extends beyond 1.5 Å in the direction perpendicular to the tilt axis.

Keywords

cryo-crinklingcryo-EMcrystalline polypeptoid membranescrystallographyspecimen flatness
Type
Software and Instrumentation
Information
Microscopy and Microanalysis , Volume 27 , Issue 4 , August 2021 , pp. 767 - 775
Check for updates
Copyright
Copyright © The Author(s), 2021. Published by Cambridge University Press on behalf of the Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

Deceased.

References

Baldwin, JM, Henderson, R, Beckman, E & Zemlin, F (1988). Images of purple membrane at 2.8 Å resolution obtained by cryo-electron microscopy. J Mol Biol 202(3), 585–591.CrossRefGoogle ScholarPubMed
Booy, FP & Pawley, JB (1993). Cryo-crinkling — What happens to carbon-films on copper grids at low-temperature. Ultramicroscopy 48(3), 273–280.CrossRefGoogle ScholarPubMed
Brilot, AF, Chen, JZ, Cheng, AC, Pan, JH, Harrison, SC, Potter, CS, Carragher, B, Henderson, R & Grigorieff, N (2012). Beam-induced motion of vitrified specimen on holey carbon film. J Struct Biol 177(3), 630–637.CrossRefGoogle ScholarPubMed
Butt, HJ, Wang, DN, Hansma, PK & Kuhlbrandt, W (1991). Effect of surface-roughness of carbon support films on high-resolution electron-diffraction of 2-dimensional protein crystals. Ultramicroscopy 36(4), 307–318.CrossRefGoogle Scholar
Calarco, PG, Peachey, LD & Williams, DB, America, E.M.S.o., Microscopy, I.C.o.E., Microscopy, I.F.o.S.f.E. & Society, M.A. (1990). Electron Microscopy 1990: Proceedings of the XIIth International Congress for Electron Microscopy Held in Seattle, Washington, USA - 48th Annual Meeting of the Electron Microscopy Society of America and 25th Annual Meeting of the Microbeam Analysis Society. Biological Sciences. San Francisco Press.Google Scholar
Cheng, YF (2015). Single-particle cryo-EM at crystallographic resolution. Cell 161(3), 450–457.CrossRefGoogle ScholarPubMed
Downing, KH (2003). Support films with uniform hole size. Microsc Today 11(5), 54–54.CrossRefGoogle Scholar
Fujiyoshi, Y (1998). The structural study of membrane proteins by electron crystallography. Adv Biophys 35, 25–80.CrossRefGoogle ScholarPubMed
Gipson, B, Zeng, X, Zhang, ZY & Stahlberg, H (2007). 2DX – user-friendly image processing for 2D crystals. J Struct Biol 157(1), 64–72.CrossRefGoogle ScholarPubMed
Glaeser, RM (1992). Specimen flatness of thin crystalline arrays — Influence of the substrate. Ultramicroscopy 46(1-4), 33–43.CrossRefGoogle ScholarPubMed
Glaeser, RM (2007). Electron Crystallography of Biological Macromolecules. Oxford, New York: Oxford University Press.Google Scholar
Glaeser, RM, McMullan, G, Faruqi, AR & Henderson, R (2011). Images of paraffin monolayer crystals with perfect contrast: Minimization of beam-induced specimen motion. Ultramicroscopy 111(2), 90–100.CrossRefGoogle ScholarPubMed
Gyobu, N, Tani, K, Hiroaki, Y, Kamegawa, A, Mitsuoka, K & Fujiyoshi, Y (2004). Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique. J Struct Biol 146(3), 325–333.CrossRefGoogle ScholarPubMed
Han, BG, Wolf, SG, Vonck, J & Glaeser, RM (1994). Specimen flatness of glucose-embedded biological-materials for electron crystallography Is affected significantly by the choice of carbon evaporation stock. Ultramicroscopy 55(1), 1–5.CrossRefGoogle ScholarPubMed
Haynes, WM (2015). Handbook of Chemistry and Physics. Boca raton, FL: CRC Press.Google Scholar
Henderson, R, Baldwin, JM, Ceska, TA, Zemlin, F, Beckmann, E & Downing, KH (1990). Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy. J Mol Biol 213(4), 899–929.CrossRefGoogle Scholar
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 a resolution. Ultramicroscopy 19(2), 147–178.CrossRefGoogle Scholar
Henderson, R & Glaeser, RM (1985). Quantitative-analysis of image-contrast in electron-micrographs of beam-sensitive crystals. Ultramicroscopy 16(2), 139–150.CrossRefGoogle Scholar
Hite, RK, Schenk, AD, Li, ZL, Cheng, YF & Walz, T (2010). Collecting electron crystallographic data of two-dimensional protein crystals. Methods Enzymol 481, 251–282.CrossRefGoogle ScholarPubMed
Jiang, X, Greer, DR, Kundu, J, Ophus, C, Minor, AM, Prendergast, D, Zuckermann, RN, Balsara, NP & Downing, KH (2018). Imaging unstained synthetic polymer crystals and defects on atomic length scales using cryogenic electron microscopy. Macromolecules 51(19), 7794–7799.CrossRefGoogle Scholar
Jiang, X, Xuan, ST, Kundu, J, Prendergast, D, Zuckermann, RN & Balsara, NP (2019). Effect of processing and end groups on the crystal structure of polypeptoids studied by cryogenic electron microscopy at atomic length scales. Soft Matter 15(23), 4723–4736.CrossRefGoogle ScholarPubMed
Kuhlbrandt, W (2014). The resolution revolution. Science 343(6178), 1443–1444.CrossRefGoogle ScholarPubMed
Kuhlbrandt, W, Wang, DN & Fujiyoshi, Y (1994). Atomic model of plant light-harvesting complex by electron crystallography. Nature 367(6464), 614–621.CrossRefGoogle ScholarPubMed
Li, XM, Mooney, P, Zheng, S, Booth, CR, Braunfeld, MB, Gubbens, S, Agard, DA & Cheng, YF (2013). Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10(6), 584.CrossRefGoogle ScholarPubMed
Mastronarde, DN (2005). Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol 152(1), 36–51.CrossRefGoogle ScholarPubMed
Miyazawa, A, Fujiyoshi, Y, Stowell, M & Unwin, N (1999). Nicotinic acetylcholine receptor at 4.6 angstrom resolution: Transverse tunnels in the channel wall. J Mol Biol 288(4), 765–786.CrossRefGoogle Scholar
Nam, KT, Shelby, SA, Choi, PH, Marciel, AB, Chen, R, Tan, L, Chu, TK, Mesch, RA, Lee, BC, Connolly, MD, Kisielowski, C & Zuckermann, RN (2010). Free-floating ultrathin two-dimensional crystals from sequence-specific peptoid polymers. Nat Mater 9(5), 454–460.CrossRefGoogle ScholarPubMed
Nogales, E, Wolf, SG, Khan, IA, Luduena, RF & Downing, KH (1995). Structure of tubulin at 6.5 angstrom and location of the taxol-binding site. Nature 375(6530), 424–427.CrossRefGoogle Scholar
Quispe, J, Damiano, J, Mick, SE, Nackashi, DP, Fellmann, D, Ajero, TG, Carragher, B & Potter, CS (2007). An improved holey carbon film for cryo-electron microscopy. Microsc Microanal 13(5), 365–371.CrossRefGoogle ScholarPubMed
Rhinow, D & Kuhlbrandt, W (2008). Electron cryo-microscopy of biological specimens on conductive titanium-silicon metal glass films. Ultramicroscopy 108(7), 698–705.CrossRefGoogle ScholarPubMed
Robertson, EJ, Oliver, GK, Qian, M, Proulx, C, Zuckermann, RN & Richmond, GL (2014). Assembly and molecular order of two-dimensional peptoid nanosheets through the oil-water interface. Proc Natl Acad Sci USA 111(37), 13284–13289.CrossRefGoogle ScholarPubMed
Russo, CJ & Passmore, LA (2014). Ultrastable gold substrates for electron cryomicroscopy. Science 346(6215), 1377–1380.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Scheres, SH (2012). RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180(3), 519–530.CrossRefGoogle ScholarPubMed
Spence, JCH, Weierstall, U, Fricke, TT, Glaeser, RM & Downing, KH (2003). Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support. J Struct Biol 144(1–2), 209–218.CrossRefGoogle ScholarPubMed
Su, M (2019). goCTF: Geometrically optimized CTF determination for single-particle cryo-EM. J Struct Biol 205(1), 22–29.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Vonck, J (1996). A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: Part of the F helix tilts in the M to N transition. Biochemistry 35(18), 5870–5878.CrossRefGoogle Scholar
Vonck, J (2000). Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography. Ultramicroscopy 85(3), 123–129.CrossRefGoogle ScholarPubMed
Walz, T, Hirai, T, Murata, K, Heymann, JB, Mitsuoka, K, Fujiyoshi, Y, Smith, BL, Agre, P & Engel, A (1997). The three-dimensional structure of aquaporin-1. Nature 387(6633), 624–627.CrossRefGoogle ScholarPubMed
Xuan, ST, Jiang, X, Spencer, RK, Li, NK, Prendergast, D, Balsara, NP & Zuckermann, RN (2019). Atomic-level engineering and imaging of polypeptoid crystal lattices. Proc Natl Acad Sci USA 116(45), 22491–22499.CrossRefGoogle ScholarPubMed
Yoshioka, C, Carragher, B & Potter, CS (2010). Cryomesh (TM): A new substrate for cryo-electron microscopy. Microsc Microanal 16(1), 43–53.CrossRefGoogle ScholarPubMed
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(4), 331–332.CrossRefGoogle ScholarPubMed
Supplementary material: File

Jiang et al. supplementary material

Jiang et al. supplementary material

Download Jiang et al. supplementary material(File)
File 1.1 MB