Xuekui Yu provided CPV data for analysis. We acknowledge funding from NIH (GM071940 and AI094386) and NSF (DBI-1338135), PG and NP receive funding from American Heart Association Western Affiliate (13POST17340020) and the NIH Biotechnology Training Program (T32GM067555), respectively.

NOTE: A general workflow of atomic model determination by cryoEM is given in Fig. 1. In the following protocol, items 1, 2 and 4 are shared in both single particle- and helical-structural determination. Item 3.1 is for single particle structures while item 3.2 is for helical structures. Therefore, one either will follow 1, 2, 3.1, 4 or 1, 2, 3.2, 4 depending on the nature of the sample being studied.
1. Grid preparation, sample preparation, and image collection
<ol>
	<li>Place commercial cry...

<ol>
	<li>Meng, X., Zhao, G., &#38; Zhang, P. Structure of HIV-1 capsid assemblies by cryo-electron microscopy and iterative helical real-space reconstruction. J Vis Exp. doi:10.3791/3041 (2011).</li>
	<li>Meyerson, J. R. et al. Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging. J Vis Exp. doi:10.3791/2770 (2011).</li>
	<li>Ge, P., &#38; Zhou, Z. H. Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches. Proc Natl Acad Sci U S A. 108, 9637-9642, doi:10.1073/pnas.1018104108 (2011).</li>
	<li>Yu, X., Ge, P., Jiang, J., Atanasov, I., &#38; Zhou, Z. H. Atomic model of CPV reveals the mechanism used by this single-shelled virus to economically carry out functions conserved in multishelled reoviruses. Structure. 19, 652-661, doi:10.1016/j.str.2011.03.003 (2011).</li>
	<li>Liu, H. et al. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks. Science. 329, 1038-1043 (2010).</li>
	<li>Zhang, X., Jin, L., Fang, Q., Hui, W. H., &#38; Zhou, Z. H. 3.3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell. 141, 472-482 (2010).</li>
	<li>Beer, M., Frank, J., Hanszen, K. J., Kellenberger, E., &#38; Williams, R. C. The possibilities and prospects of obtaining high-resolution information (below 30 A) on biological material using the electron microscope. Some comments and reports inspired by an EMBO workshop held at Gais, Switzerland, October 1973. Q Rev Biophys. 7, 211-238 (1974).</li>
	<li>Bharat, T. A. M. et al. Structure of the immature retroviral capsid at 8[thinsp]A resolution by cryo-electron microscopy. Nature. 487, 385-389, doi:10.1038/nature11169 (2012).</li>
	<li>Li, S., Hill, C. P., Sundquist, W. I., &#38; Finch, J. T. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature. 407, 409-413, doi:10.1038/35030177 (2000).</li>
	<li>Kimura, Y. et al. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature. 389, 206-211, doi:10.1038/38323 (1997).</li>
	<li>Nogales, E., Wolf, S. G., &#38; Downing, K. H. Structure of the alpha beta tubulin dimer by electron crystallography. Nature. 391, 199-203, doi:10.1038/34465 (1998).</li>
	<li>Yonekura, K., Maki-Yonekura, S., &#38; Namba, K. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 424, 643-650, doi:10.1038/nature01830 (2003).</li>
	<li>Zhang, X. et al. Structure of Sputnik, a virophage, at 3.5-A resolution. Proc Natl Acad Sci U S A. 109, 18431-18436, doi:10.1073/pnas.1211702109 (2012).</li>
	<li>Ludtke, S. J. et al. De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure. 16, 441-448, doi:10.1016/j.str.2008.02.007 (2008).</li>
	<li>Grigorieff, N., &#38; Harrison, S. C. Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol. 21, 265-273, doi:10.1016/j.sbi.2011.01.008 (2011).</li>
	<li>Zhou, Z. H. Atomic resolution cryo electron microscopy of macromolecular complexes. Adv Protein Chem Struct Biol. 82, 1-35, doi:10.1016/B978-0-12-386507-6.00001-4 (2011).</li>
	<li>Zhou, Z. H. Structures of viral membrane proteins by high-resolution cryoEM. Curr Opin Virol. 5, 111-119, doi:10.1016/j.coviro.2014.04.001 (2014).</li>
	<li>Mindell, J. A., &#38; Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol. 142, 334-347 (2003).</li>
	<li>Ludtke, S. J., Baldwin, P. R., &#38; Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol. 128, 82-97, doi:10.1006/jsbi.1999.4174 (1999).</li>
	<li>Liang, Y., Ke, E. Y., &#38; Zhou, Z. H. IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database. J Struct Biol. 137, 292-304 (2002).</li>
	<li>Zhang, X., &#38; Zhou, Z. H. Low cost, high performance GPU computing solution for atomic resolution cryoEM single-particle reconstruction. J Struct Biol. 172, 400-406, doi:10.1016/j.jsb.2010.05.006 (2010).</li>
	<li>De Rosier, D. J., &#38; Klug, A. Reconstruction of Three Dimensional Structures from Electron Micrographs. Nature. 217, 130-134 (1968).</li>
	<li>Egelman, E. H. The iterative helical real space reconstruction method: surmounting the problems posed by real polymers. J Struct Biol. 157, 83-94, doi:S1047-8477(06)00168-7 [pii]10.1016/j.jsb.2006.05.015 (2007).</li>
	<li>Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nat. Protocols. 2, 2728-2733, doi:10.1038/nprot.2007.406 (2007).</li>
	<li>Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 66, 213-221, doi:10.1107/S0907444909052925 (2010).</li>
	<li>Zhang, J. et al. Mechanism of folding chamber closure in a group II chaperonin. Nature. 463, 379-383, doi:10.1038/nature08701 (2010).</li>
	<li>Emsley, P., Lohkamp, B., Scott, W. G., &#38; Cowtan, K. Features and development of Coot. Acta Crystallographica Section D. 66, 486-501, doi:10.1107/S0907444910007493 (2010).</li>
	<li>Li, Y., &#38; Zhang, Y. REMO: A new protocol to refine full atomic protein models from C-alpha traces by optimizing hydrogen-bonding networks. Proteins: Structure, Function, and Bioinformatics. 76, 665-676, doi:10.1002/prot.22380 (2009).</li>
	<li>Zhang, X., &#38; Zhou, Z. H. Limiting factors in atomic resolution cryo electron microscopy: no simple tricks. J Struct Biol. 175, 253-263, doi:10.1016/j.jsb.2011.05.004 (2011).</li>
	<li>Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol. 157, 38-46, doi:10.1016/j.jsb.2006.05.009 (2007).</li>
</ol>

With advances in imaging hardware and software, cryoEM has come of age as a structural biology method enabling de novo atomic models based on density maps alone. However, some major limitations persist for this technique, in particular the limitations in sample types and preparations which are suitable for use in cryoEM. Thus when considering cryoEM for structural determination, the most important question to ask is still: is this the right technique for my sample 15-17? While X-ray crystallography an...

Beginning in the early 1970s, electron microscopy has been used in structural biology to investigate macromolecular assemblies at a &#8220;biologically significant&#8221; resolution 7. Atomic resolution protein structures determined by x-ray crystallography or NMR were docked into the lower resolution (30-8 &#197;) electron microscopy density maps (for example8,9). In this way, interactions between subunits of a large complex could be assessed to give hints at the overall function of a macromolecular assembly. While this approach is still frequently used for complexes with low or no symmetry, advances over the last twenty years in sample preparation, microscope design, data collection, and data processing have made de novo atomic model building, based on the EM density map alone, possible for certain types of samples 3,6.
Three broad types of sample are suitable for de nove atomic model building by cryoEM: 2D crystalline arrays, such as bacteriorhodopsin 10 and tubulin 11, helical assemblies, such as tobacco mosaic virus 3 and the bacterial flagellum 12, and complexes with high symmetry, such as icosahedral viruses 4-6,13 and GroEL 14. Our laboratory has been successful in deriving several atomic models for helical assemblies and icosahedral viruses 3,5,6. Based on past experiences we present the current article, which is intended to highlight some essential steps which allow for the electron density map to be refined to 3-4 &#197; resolution.
Below, we provide a working protocol with detailed steps for determining icosahedral and helical structures to about 3-&#197; resolution and for deriving atomic models. We also provide representative results and a discussion focusing on crucial steps in sample preparation and image processing for achieving atomic resolution structures. For in-depth understanding of the underlying theory and recent progress, viewers are advised to read recent reviews on this topic 15-17.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Name of Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:96px;">Catalog Number</td>
			<td style="width:97px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitrobot</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Titan Krios</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quantifoil cryoEM grids</td>
			<td>Quantifoil microtools GmbH</td>
			<td></td>
			<td>http://www.quantifoil.com/qfstr_en.php4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1,2-dichloro-ethane</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;107-06-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SO163 Films and developer/fixer</td>
			<td>Kodak</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Super CoolScan 9000 ED</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMAN 1,2</td>
			<td>Baylor College of Medicine</td>
			<td></td>
			<td>http://blake.bcm.edu/emanwiki/EMAN/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Linux cluster with scientific linux (redhat enterprise linux)</td>
			<td>Redhat / Fermilab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chimera</td>
			<td>UCSF</td>
			<td></td>
			<td>http://www.cgl.ucsf.edu/chimera/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coot</td>
			<td></td>
			<td></td>
			<td>http://lmb.bioch.ox.ac.uk/coot/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CNS</td>
			<td></td>
			<td></td>
			<td>http://cns-online.org/v1.3/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phenix</td>
			<td></td>
			<td></td>
			<td>http://www.phenix-online.org/</td>
		</tr>
	</tbody>
</table>

deriving de novo atomic models by cryo electron microscopy

Cryo electron microscopy is a structural biology technique for determining three-dimensional structures of supramolecular complexes in solution. In cryoEM, samples in their physiological, non-crystalline state are embedded in vitreous ice during low dose imaging. A three-dimensional density map of the sample is obtained by combining tens of thousands of cryoEM images. In recent years, the resolution of cryoEM has improved steadily, reaching a resolution sufficient for building atomic models from the cryoEM density map alone.
In this video article, we illustrate how such atomic models can be derived by cryoEM. Others have described the detailed procedures for preparing frozen hydrated samples, image acquisition and the basic steps of data processing of cryoEM 1,2. We will focus on several criticle technical aspects important for reaching 3-4 &#197; resolution and for building reliable atomic models. We use the tobacco mosaic virus (TMV) and cytoplasmic polyhedrosis virus (CPV) as representative complexes with helical and icosahedral objects, respectively. De novo atomic models of these objects have previously been determined by cryoEM and published, and their biological relevance is described in previous publications3-6.

The steps of cryoEM structure determination include sample purification and vitrification, low-dose imaging, orientation determination and refinement, 3D reconstruction, and atomic model building. First, samples particularly suitable for high-resolution cryoEM analysis are those with adequate size (&#62;1MDa molecular weight, &#60; 150 nm in thickness) such that sufficient contrast for visualization is provided, and with structural uniformity and integrity such that all particles are structurally identical for averaging....

Deriving De Novo Atomic Models By Cryo Electron Microscopy

Cryo electron microscopy (cryoEM) can be employed to derive de novo atomic models of macromolecular complexes in solution. The steps involved in high resolution cryoEM of biological molecules, from image recording, to data processing, to atomic modeling based on the resulting cryoEM density map, are illustrated.

Deriving De Novo Atomic Models by Cryo Electron Microscopy

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Deriving <em>De Novo</em> Atomic Models by Cryo Electron Microscopy

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