Abstract
Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.
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References
Zernike F (1955) How I discovered phase contrast. Science 121:345–349
Herzik MA, Wu M, Lander GC (2019) High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nat Commun 10:1032
Orlov I, Rochel N, Moras D et al (2012) Structure of the full human RXR/VDR nuclear receptor heterodimer complex with its DR3 target DNA. EMBO J 31:291–300
Dubochet J, Adrian M, Chang JJ et al (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21:129–228
Cambie R, Downing KH, Typke D et al (2007) Design of a microfabricated, two-electrode phase-contrast element suitable for electron microscopy. Ultramicroscopy 107:329–339
Danev R, Nagayama K (2001) Transmission electron microscopy with Zernike phase plate. Ultramicroscopy 88:243–252
Frindt N, Oster M, Hettler S et al (2014) In-focus electrostatic Zach phase plate imaging for transmission electron microscopy with tunable phase contrast of frozen hydrated biological samples. Microsc Microanal 20:175–183
Majorovits E, Barton B, Schultheiss K et al (2007) Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate. Ultramicroscopy 107:213–226
Walter A, Steltenkamp S, Schmitz S et al (2015) Towards an optimum design for electrostatic phase plates. Ultramicroscopy 153:22–31
Danev R, Buijsse B, Khoshouei M et al (2014) Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc Natl Acad Sci U S A 111:15635–15640
Danev R, Tegunov D, Baumeister W (2017) Using the Volta phase plate with defocus for cryo-EM single particle analysis. eLife 6:e23006
Dai W, Fu C, Raytcheva D et al (2013) Visualizing virus assembly intermediates inside marine cyanobacteria. Nature 502:707–710
Murata K, Liu X, Danev R et al (2010) Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and sub-nanometer resolutions. Structure 1993(18):903–912
Li K, Sun C, Klose T et al (2019) Sub-3 Å apoferritin structure determined with full range of phase shifts using a single position of Volta phase plate. J Struct Biol 206:225–232
Fan X, Zhao L, Liu C et al (2017) Near-atomic resolution structure determination in over-focus with Volta phase plate by Cs-corrected Cryo-EM. Structure 25:1623–1630. e3
von Loeffelholz O, Papai G, Danev R et al (2018) Volta phase plate data collection facilitates image processing and cryo-EM structure determination. J Struct Biol 202:191–199
Fan X, Wang J, Zhang X et al (2019) Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Ångstrom resolution. Nat Commun 10:2386
Hill CH, Boreikaitė V, Kumar A et al (2019) Activation of the endonuclease that defines mRNA 3’ ends requires incorporation into an 8-subunit Core cleavage and polyadenylation factor complex. Mol Cell 73:1217–1231
Khoshouei M, Radjainia M, Baumeister W et al (2017) Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate. Nat Commun 8:16099
Abeyrathne PD, Koh CS, Grant T et al (2016) Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. eLife 5:e14874
Klaholz BP, Myasnikov AG, van Heel M (2004) Visualization of release factor 3 on the ribosome during termination of protein synthesis. Nature 427:862–865
Penczek PA, Frank J, Spahn CM (2006) A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J Struct Biol 154:184–194
Scheres SH (2010) Classification of structural heterogeneity by maximum-likelihood methods. Methods Enzymol 482:295–320
von Loeffelholz O, Natchiar SK, Djabeur N et al (2017) Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes. Curr Opin Struct Biol 46:140–148
Zivanov J, Nakane T, Forsberg BO et al (2018) New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7:e42166
Afonine PV, Klaholz BP, Moriarty NW et al (2018) New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr Sect Struct Biol 74:814–840
Khoshouei M, Pfeffer S, Baumeister W et al (2017) Subtomogram analysis using the Volta phase plate. J Struct Biol 197:94–101
Rast A, Schaffer M, Albert S et al (2019) Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nat Plants 5:436
Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51
Li X, Mooney P, Zheng S et al (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590
Zheng SQ, Palovcak E, Armache JP et al (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331–332
Tegunov D, Cramer P (2019) Real-time cryo-EM data pre-processing with Warp. Nat Methods 16:1146–1152
Zhang K (2016) Gctf: real-time CTF determination and correction. J Struct Biol 193:1–12
Rohou A, Grigorieff N (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct Biol 192:216–221
Acknowledgments
We thank Jonathan Michalon, Mathieu Schaeffer, Remy Fritz, and Romaric David for IT support. This work was supported by CNRS, Association pour la Recherche sur le Cancer (ARC), Institut National du Cancer (INCa), the Fondation pour la Recherche Médicale (FRM), Ligue nationale contre le cancer (Ligue), Agence National pour la Recherche (ANR), and USIAS (USIAS-2018-012). The electron microscope facility was supported by the Alsace Region, FRM, Inserm, CNRS and ARC, the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01, by Instruct-ERIC and Instruct-ULTRA (Coordination and Support Action Number ID 731005) funded by the EU H2020 framework to further develop the services of Instruct-ERIC.
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von Loeffelholz, O., Klaholz, B.P. (2021). Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection. In: Owens, R.J. (eds) Structural Proteomics. Methods in Molecular Biology, vol 2305. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1406-8_14
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DOI: https://doi.org/10.1007/978-1-0716-1406-8_14
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