Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Real-Time Measurement of the Liquid Amount in Cryo-Electron Microscopy Grids Using Laser Diffraction of Regular 2-D Holes of the Grids

  • Ahn, Jinsook (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University) ;
  • Lee, Dukwon (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University) ;
  • Jo, Inseong (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University) ;
  • Jeong, Hyeongseop (Korea Basic Science Institute) ;
  • Hyun, Jae-Kyung (Korea Basic Science Institute) ;
  • Woo, Jae-Sung (Department of Life Sciences, Korea University) ;
  • Choi, Sang-Ho (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University) ;
  • Ha, Nam-Chul (Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University)
  • Received : 2019.10.21
  • Accepted : 2019.12.10
  • Published : 2020.03.31
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Abstract

Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine high-resolution protein structures using the limited resource of cryo-EM instrument access.

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References

  1. Arnold, S.A., Albiez, S., Bieri, A., Syntychaki, A., Adaixo, R., McLeod, R.A., Goldie, K.N., Stahlberg, H., and 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, 220-226. https://doi.org/10.1016/j.jsb.2016.11.002
  2. Bai, X.C., McMullan, G., and Scheres, S.H. (2015). How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40, 49-57. https://doi.org/10.1016/j.tibs.2014.10.005
  3. Binshtein, E. and Ohi, M.D. (2015). Cryo-electron microscopy and the amazing race to atomic resolution. Biochemistry 54, 3133-3141. https://doi.org/10.1021/acs.biochem.5b00114
  4. Feng, X., Fu, Z., Kaledhonkar, S., Jia, Y., Shah, B., Jin, A., Liu, Z., Sun, M., Chen, B., and Grassucci, R.A. (2017). A fast and effective microfluidic sprayingplunging method for high-resolution single-particle cryo-EM. Structure 25, 663-670.e3. https://doi.org/10.1016/j.str.2017.02.005
  5. Glaeser, R.M. (2008). Retrospective: radiation damage and its associated "information limitations". J. Struct. Biol. 163, 271-276. https://doi.org/10.1016/j.jsb.2008.06.001
  6. Glaeser, R.M., Han, B.G., Csencsits, R., Killilea, A., Pulk, A., and Cate, J.H. (2016). Factors that influence the formation and stability of thin, cryo-EM specimens. Biophys. J. 110, 749-755. https://doi.org/10.1016/j.bpj.2015.07.050
  7. Jain, T., Sheehan, P., Crum, J., Carragher, B., and Potter, C.S. (2012). Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68-75. https://doi.org/10.1016/j.jsb.2012.04.020
  8. Kimanius, D., Forsberg, B.O., Scheres, S.H., and Lindahl, E. (2016). Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife 5, e18722. https://doi.org/10.7554/eLife.18722
  9. Liu, Y. and Sigworth, F.J. (2014). Automatic cryo-EM particle selection for membrane proteins in spherical liposomes. J. Struct. Biol. 185, 295-302. https://doi.org/10.1016/j.jsb.2014.01.004
  10. Massover, W.H. (2011). New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation. Micron 42, 141-151. https://doi.org/10.1016/j.micron.2010.05.006
  11. McMullan, G., Faruqi, A.R., Clare, D., and Henderson, R. (2014). Comparison of optimal performance at 300 keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147, 156-163. https://doi.org/10.1016/j.ultramic.2014.08.002
  12. Noble, A.J., Dandey, V.P., Wei, H., Brasch, J., Chase, J., Acharya, P., Tan, Y.Z., Zhang, Z., Kim, L.Y., Scapin, G., et al. (2018). Routine single particle CryoEM sample and grid characterization by tomography. Elife 7, e34257. https://doi.org/10.7554/eLife.34257
  13. Razinkov, I., Dandey, V., Wei, H., Zhang, Z., Melnekoff, D., Rice, W.J., Wigge, C., Potter, C.S., and Carragher, B. (2016). A new method for vitrifying samples for cryoEM. J. Struct. Biol. 195, 190-198. https://doi.org/10.1016/j.jsb.2016.06.001
  14. Rubinstein, J.L., Guo, H., Ripstein, Z.A., Haydaroglu, A., Au, A., Yip, C.M., Di Trani, J.M., Benlekbir, S., and Kwok, T. (2019). Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device. Acta Crystallogr. D Struct. Biol. 75, 1063-1070. https://doi.org/10.1107/S2059798319014372
  15. Scheres, S.H. (2012). RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519-530. https://doi.org/10.1016/j.jsb.2012.09.006
  16. Stagg, S.M., Lander, G.C., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D., Mallick, S.P., Avila, R.M., Carragher, B., and Potter, C.S. (2006). Automated cryoEM data acquisition and analysis of 284742 particles of GroEL. J. Struct. Biol. 155, 470-481. https://doi.org/10.1016/j.jsb.2006.04.005