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Slideshow

Recent Developments in Sample Preparation for Single Particle Cryo-EM

Chemistry Building, Room 400
Analytical Seminar

Single particle electron cryomicroscopy (cryo-EM) produces 3D structures of macromolecules by combining many EM images of particles embedded in a thin layer of vitreous ice.  A new generation of direct electron detectors has enabled the production of atomic-resolution structures of biomolecules.1  Despite the impressive gains in resolution, structural determination by cryo-EM remains a slow process involving considerable trial and error to obtain a sample of sufficient quality.2  The traditional workflow involves pipetting sample on an amorphous carbon grid, blotting the sample to create a thin film, and then plunging the grid into a cryogenic bath to flash-freeze the sample while preserving its native structure.  Recent research in sample preparation aims to reduce sample motion, help control ice thickness, and creating preferred sample orientation.  The use of a gold sample support greatly reduces electron-beam induced particle motion, which leads to higher quality images.3  A microfluidic method for depositing sample onto the EM grid allows for better control of ice thickness than the traditional blotting approach.4  Lastly, the use of protein cages created by DNA origami shows promise for allowing control of sample orientation.5  

1.         Orlova, E. V.; Saibil, H. R., Structural Analysis of Macromolecular Assemblies by Electron Microscopy. Chemical Reviews 2011, 111 (12), 7710-7748.

2.         Cheng, Y.; Grigorieff, N.; Penczek, Pawel A.; Walz, T., A Primer to Single-Particle Cryo-Electron Microscopy. Cell 2015, 161 (3), 438-449.

3.         Russo, C. J.; Passmore, L. A., Ultrastable gold substrates for electron cryomicroscopy. Science 2014, 346 (6215), 1377-1380.

4.         Feng, X.; Fu, Z.; Kaledhonkar, S.; Jia, Y.; Shah, B.; Jin, A.; Liu, Z.; Sun, M.; Chen, B.; Grassucci, R. A.; Ren, Y.; Jiang, H.; Frank, J.; Lin, Q., A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM. Structure 2017, 25 (4), 663-670.e3.

5.         Martin, T. G.; Bharat, T. A. M.; Joerger, A. C.; Bai, X.-c.; Praetorius, F.; Fersht, A. R.; Dietz, H.; Scheres, S. H. W., Design of a molecular support for cryo-EM structure determination. Proceedings of the National Academy of Sciences 2016, 113 (47), E7456-E7463.

 

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