This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol><li>Nickell, S., Mihalache, O., Beck, F., Hegerl, R., Korinek, A., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+analysis+of+the+26S+proteasome+by+cryoelectron+tomography%5BTitle%5D)+AND+%22Biochemical+and+biophysical+research+communications%22%5BJournal%5D)">Structural analysis of the 26S proteasome by cryoelectron tomography.</a> Biochemical and biophysical research communications. 353 (1), 115-120 (2007).</li>
<li>Goldie, K. N., Wedig, T., Mitra, A. K., Aebi, U., Herrmann, H., Hoenger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+3-D+structure+of+vimentin+intermediate+filaments+by+cryo-electron+tomography%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography.</a> Journal of structural biology. 158 (3), 378-385 (2007).</li>
<li>Cheng, Y., Boll, W., Kirchhausen, T., Harrison, S. C., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomography+of+clathrin-coated+vesicles%3A+structural+implications+for+coat+assembly%5BTitle%5D)+AND+%22Journal+of+molecular+biology%22%5BJournal%5D)">Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly.</a> Journal of molecular biology. 365 (3), (2007).</li>
<li>Grünewald, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+structure+of+herpes+simplex+virus+from+cryo-electron+tomography%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Three-dimensional structure of herpes simplex virus from cryo-electron tomography.</a> Science. 302 (5649), 1396-1398 (2003).</li>
<li>Murphy, G. E., Leadbetter, J. R., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+structure+of+the+complete+Treponema+primitia+flagellar+motor%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In situ structure of the complete Treponema primitia flagellar motor.</a> Nature. 442 (7106), 1062-1064 (2006).</li>
<li>Medalia, O., Weber, I., Frangakis, A., Nicastro, D., Gerisch, G., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+architecture+in+eukaryotic+cells+visualized+by+cryoelectron+tomography%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.</a> Science. 298 (5596), 1209-1213 (2002).</li>
<li>Heymann, J. B., Cardone, G., Winkler, D. C., Steven, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+resources+for+cryo-electron+tomography+in+Bsoft%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Computational resources for cryo-electron tomography in Bsoft.</a> Journal of structural biology. 161 (3), 232-242 (2008).</li>
<li>Kremer, J., Mastronarde, D., McIntosh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer+visualization+of+three-dimensional+image+data+using+IMOD%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Computer visualization of three-dimensional image data using IMOD.</a> Journal of structural biology. 116 (1), 71-76 (1996).</li>
<li>Castaño-Díez, D., Kudryashev, M., Arheit, M., Stahlberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamo%3A+a+flexible%2C+user-friendly+development+tool+for+subtomogram+averaging+of+cryo-EM+data+in+high-performance+computing+environments%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.</a> Journal of structural biology. 178 (2), 139-151 (2012).</li>
<li>Hrabe, T., Chen, Y., Pfeffer, S., Cuellar, L. K., Mangold, A. -V., Förster, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PyTom%3A+a+python-based+toolbox+for+localization+of+macromolecules+in+cryo-electron+tomograms+and+subtomogram+analysis%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis.</a> Journal of structural biology. 178 (2), 177-188 (2012).</li>
<li>Fernández, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+methods+for+electron+tomography%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Computational methods for electron tomography.</a> 43 (10), Micron (Oxford, England). 1010-1030 (2012).</li>
<li>Briggs, J. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+biology+in+situ--the+potential+of+subtomogram+averaging%29%5BTitle%5D)+AND+%22Current+opinion+in+structural+biology%22%5BJournal%5D)">Structural biology in situ--the potential of subtomogram averaging).</a> Current opinion in structural biology. 23 (2), 261-267 (2013).</li>
<li>Frangakis, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+macromolecular+complexes+in+cryoelectron+tomograms+of+phantom+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Identification of macromolecular complexes in cryoelectron tomograms of phantom cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (22), 14153-14158 (2002).</li>
<li>Zanetti, G., Briggs, J. A. G., Grünewald, K., Sattentau, Q. J., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-electron+tomographic+structure+of+an+immunodeficiency+virus+envelope+complex+in+situ%5BTitle%5D)+AND+%22PLoS+pathogens%22%5BJournal%5D)">Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ.</a> PLoS pathogens. 2 (8), (2006).</li>
<li>Liu, J., Bartesaghi, A., Borgnia, M. J., Sapiro, G., Subramaniam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+architecture+of+native+HIV-1+gp120+trimers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Molecular architecture of native HIV-1 gp120 trimers.</a> Nature. 455 (7209), (2008).</li>
<li>Meyerson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+molecular+structures+of+HIV+envelope+glycoproteins+using+cryo-electron+tomography+and+automated+sub-tomogram+averaging%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging.</a> Journal of visualized experiments : JoVE. (58), (2011).</li>
<li>Baker, T. S., Olson, N. H., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adding+the+third+dimension+to+virus+life+cycles%3A+three-dimensional+reconstruction+of+icosahedral+viruses+from+cryo-electron+micrographs%5BTitle%5D)+AND+%22Microbiology+and+molecular+biology+reviews+%3A+MMBR%22%5BJournal%5D)">Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs.</a> Microbiology and molecular biology reviews : MMBR. 63 (4), 862-922 (1999).</li>
<li>Huiskonen, J. T., Butcher, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane-containing+viruses+with+icosahedrally+symmetric+capsids%5BTitle%5D)+AND+%22Current+opinion+in+structural+biology%22%5BJournal%5D)">Membrane-containing viruses with icosahedrally symmetric capsids.</a> Current opinion in structural biology. 17 (2), 229-236 (2007).</li>
<li>Liljeroos, L., Huiskonen, J. T., Ora, A., Susi, P., Butcher, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+cryotomography+of+measles+virus+reveals+how+matrix+protein+coats+the+ribonucleocapsid+within+intact+virions%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (44), (2011).</li>
<li>Arranz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+native+influenza+virion+ribonucleoproteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The structure of native influenza virion ribonucleoproteins.</a> Science. 338 (6114), 1634-1637 (2012).</li>
<li>Karotki, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eisosome+proteins+assemble+into+a+membrane+scaffold%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Eisosome proteins assemble into a membrane scaffold.</a> Journal of Cell Biology. 195 (5), 889-902 (2011).</li>
<li>Pietilä, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Virion+architecture+unifies+globally+distributed+pleolipoviruses+infecting+halophilic+archaea%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">Virion architecture unifies globally distributed pleolipoviruses infecting halophilic archaea.</a> Journal of virology. 86 (9), 5067-5079 (2012).</li>
<li>Huiskonen, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+cryotomography+of+Tula+hantavirus+suggests+a+unique+assembly+paradigm+for+enveloped+viruses%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses.</a> Journal of virology. 84 (10), 4889-4897 (2010).</li>
<li>Bowden, T. A., Bitto, D., McLees, A., Yeromonahos, C., Elliott, R. M., Huiskonen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthobunyavirus+ultrastructure+and+the+curious+tripodal+glycoprotein+spike%5BTitle%5D)+AND+%22PLoS+pathogens%22%5BJournal%5D)">Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.</a> PLoS pathogens. 9 (5), (2013).</li>
<li>Maurer, U. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Structure+of+Herpesvirus+Fusion+Glycoprotein+B-Bilayer+Complex+Reveals+the+Protein-Membrane+and+Lateral+Protein-Protein+Interaction%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">The Structure of Herpesvirus Fusion Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction.</a> Structure. 21 (8), London, England. 1396-1405 (1993).</li>
<li>Gan, L., Ladinsky, M. S., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+in+a+marine+picoeukaryote+is+a+disordered+assemblage+of+nucleosomes%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">Chromatin in a marine picoeukaryote is a disordered assemblage of nucleosomes.</a> Chromosoma. 122 (5), 377-386 (2013).</li>
<li>Scheres, S. H. W., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevention+of+overfitting+in+cryo-EM+structure+determination%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Prevention of overfitting in cryo-EM structure determination.</a> Nature. 9 (9), 853-854 (2012).</li>
<li>Heymann, J. B., Belnap, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bsoft%3A+image+processing+and+molecular+modeling+for+electron+microscopy%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Bsoft: image processing and molecular modeling for electron microscopy.</a> Journal of structural biology. 157 (1), 3-18 (2007).</li>
<li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+density+maps+with+UCSF+Chimera%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">Visualizing density maps with UCSF Chimera.</a> Journal of structural biology. 157 (1), 281-287 (2007).</li>
<li>Faini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structures+of+COPI-coated+vesicles+reveal+alternate+coatomer+conformations+and+interactions%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions.</a> Science. 336 (6087), 1451-1454 (2012).</li>
<li>Wu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabs+enable+single+particle+cryoEM+studies+of+small+proteins%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Fabs enable single particle cryoEM studies of small proteins.</a> Structure. 20 (4), London, England. 582-592 (2012).</li>
</ol>

The authors have nothing to disclose.

Knowledge of viral glycoprotein spike structure on the virion membrane is essential for understanding virus replication and developing therapeutics to treat and prevent infection. Electron cryo-microscopy combined with single particle averaging has emerged as the most utilized method to solve the structures of enveloped virus particles, including the glycoprotein spikes. However, this method is limited to icosahedrally symmetric viruses. Here, through the application of electron cryo-tomography and subtomogram averaging in Jsubtomo, we have outlined a general protocol for determining glycoprotein spikes on pleomorphic enveloped viruses that are not amenable to other current structural biology methods. Our representative results demonstrate that the resolution of this method is sufficient to reveal insights into domain architecture, oligomerization, and higher order organization of glycoprotein spikes on intact virions.

The most critical step within this protocol is constructing two reliable starting models that are statistically independent from one another. Successful execution of this step assumes that glycoprotein spikes are sufficiently large and not packed against each other too tightly, so that individual spikes can be visually recognized and manually picked in the tomograms, and two independent models averaged. If this is not feasible, two modifications to the protocol can be attempted. First, two independent random models can be constructed by first defining two random subsets of subtomograms and then averaging the subtomograms within these subsets30. Second, if a structure of the isolated spike has been derived by other means, for example by X-ray crystallography, it can be used as a starting model. However, care must be taken to low-pass filter this model using a low-resolution cut-off (50-70 &#197;), as the two resulting models in the next round of refinement will be statistically independent only beyond this resolution. Due to this caveat, the former approach is recommended.

The obtainable resolution from this protocol depends on four major factors: i. data collection strategy and the quality of the input data, ii. number of the subtomograms, iii. alignment accuracy of the subtomograms, and iv. heterogeneity of the structures. While the first and second limitation can be largely overcome by using high signal-to-noise direct electron detectors combined with CTF corrected tomography and automated data collection, the alignment accuracy is further affected by the size and shape of the structure of interest itself. When applying this protocol on small spikes lacking prominent features, it may be advantageous to bind Fab fragments to the spike to improve the alignment accuracy and thus resolution31. Finally, if the structures to be averaged exhibit multiple conformations, sub-tomogram classification methods may be used to average different conformations separately. To that end, Jsubtomo integrates with the Dynamo package, offering powerful subtomogram classification9.

The above protocol is complementary to X-ray crystallography of isolated viral glycoproteins. Crystallographic structures can be fitted into sub-tomogram averages to obtain the precise orientation of the glycoprotein with respect to the virion membrane. Application of this methodology will undoubtedly continue to shed light onto enveloped virus structure and pathobiology.

Electron cryo-tomography is an electron cryo-microscopy imaging technique allowing the calculation of a three-dimensional (3D) reconstruction of complex biological specimens. Suitable specimens range from purified macromolecular complexes1, filaments2, coated vesicles3, and pleomorphic membrane viruses4 to whole prokaryotic cells5 and even thin areas of whole eukaryotic cells6. Following the data collection of a tilt-series, 3D tomographic volumes, or tomograms, may be calculated using several established software packages, including Bsoft7 and IMOD8.
Two aspects inherent to the study of biological specimens by electron cryo-tomography limit the biological interpretation of the corresponding tomographic volumes. First, due to the limited electron dose that can be applied to biological materials before introducing significant radiation damage, signal-to-noise ratios in tomographic data are typically very low. Second, as a result of limited sample tilt geometry during data collection, some views of the object remain absent, leading to a so-called &#8216;missing wedge&#8217; artifact in the tomographic volume. However, both of these limitations can be overcome if the tomographic volume contains repeating identical structures, such as macromolecular complexes, that can be successfully averaged9-12.
Prior to averaging structures from tomogram reconstructions, objects of interest must be found and aligned to the same orientation. Locating such structures may be achieved by cross-correlation of a template structure in the tomographic volume using an approach often referred to as template matching13. The template used in this matching process can be derived from electron cryo-microscopy or electron cryo-tomography combined with 3D reconstruction, or it can be a density map simulated from an atomic structure. Several computational packages have been developed to carry out these tasks11.
Averaging of glycoprotein spikes of membrane viruses, such as HIV-1, has been a particularly successful approach for studying their structure14-16. An understanding of the structure is integral for revealing both the molecular basis of virus&#8211;host interactions and guiding antiviral and vaccine design development. While macromolecular crystallography is the technique of choice for high-resolution (usually better than 4 &#197;) structural analysis of individual viral glycoproteins and their complexes, the X-ray structures resulting from this method are of proteins isolated from the natural membranous environment on the virion. Thus, important details such as the higher order architecture of viral glycoproteins, in the context of the virion, remain lacking. On the other hand, electron cryo-microscopy and single particle reconstruction of entire enveloped viruses is restricted to virions with icosahedral symmetry17,18. Electron cryotomography combined with sub-volume alignment has thus emerged as a complementary technique allowing the study of glycoprotein spikes of irregularly shaped, pleomorphic viruses in situ.
We have developed software named Jsubtomo (www.opic.ox.ac.uk/jsubtomo) for the detection, alignment, and averaging of tomographic sub-volumes. Jsubtomo has been utilized in the structure determination of a number of cellular and viral structures19-26. Here, we outline a detailed protocol, which enables the determination viral-surface spike structures. To circumvent over-refinement of averaged structures by correlating noise, the &#8216;gold-standard&#8217; refinement scheme is adopted10,27. Finally, strategies for visualization and interpretation of typical results are discussed.

<table><tbody><tr><td>Jsubtomo (ver 1.3.1)</td><td>University of Oxford</td><td>n/a</td><td>www.opic.ac.uk/jsubtomo</td></tr><tr><td>Bsoft (ver 1.8.7)</td><td>NIAMS, NIH</td><td>n/a</td><td>bsoft.ws</td></tr><tr><td>UCSF Chimera</td><td>UCSF</td><td>n/a</td><td>www.cgl.ucsf.edu/chimera</td></tr></tbody></table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

We demonstrate the application of the sub-tomogram averaging workflow outlined above for the envelope glycoprotein complex of Bunyamwera virus (Orthobunyavirus, Bunyaviridae) using a previously published data set24. Data collection and refinement parameters are listed in Table 1. One representative tomogram is shown in Figure 2.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Parameter (unit)</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Data collection</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltage (kV)</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Calibrated magnification (X)</td>
			<td>111,000</td>
		</tr>
		<tr>
			<td>Pixel size (&#197;)</td>
			<td>5.4</td>
		</tr>
		<tr>
			<td>Estimated dose (e&#8211;/ &#197;2)</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Underfocus (&#956;m)</td>
			<td>4.0&#8211;4.5</td>
		</tr>
		<tr>
			<td>First CTF zero (&#197;)a</td>
			<td>26&#8211;30</td>
		</tr>
		<tr>
			<td>Tilt range (&#176;)</td>
			<td>&#8211;60&#8211;60</td>
		</tr>
		<tr>
			<td>Tilt sampling (&#176;)</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Data and refinement</td>
			<td></td>
		</tr>
		<tr>
			<td>Tomograms</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Virions</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Seeds per virion</td>
			<td>106</td>
		</tr>
		<tr>
			<td>Total number of seeds</td>
			<td>3,074</td>
		</tr>
		<tr>
			<td>Spikes detected</td>
			<td>1,346</td>
		</tr>
		<tr>
			<td>Spikes after removing overlaps</td>
			<td>1,401</td>
		</tr>
		<tr>
			<td>Spikes after cross-correlation based selection</td>
			<td>1,022</td>
		</tr>
		<tr>
			<td>Spikes included in the final average</td>
			<td>1,022</td>
		</tr>
		<tr>
			<td>Symmetry</td>
			<td>C3</td>
		</tr>
		<tr>
			<td>Angular sampling (&#176;)</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Resolution range used in the refinement (&#197;)</td>
			<td>42&#8211;334</td>
		</tr>
		<tr>
			<td>Final resolution estimate (&#197;)b</td>
			<td>35</td>
		</tr>
	</tbody>
</table>
Table 1: Bunyamwera data collection and refinement statistics. 
a CTF, contrast transfer function. 
b Calculated using Fourier shell correlation between two independently refined structures at a threshold of 0.143.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 
Figure 2: Slice through a tomogram of Bunyamwera virions. Several spike side views evident in the periphery of each virion are indicated with arrowheads. The tomogram has been low-pass filtered to 60 &#197;. Scale bar 100 nm.
First, we refined an initial model using 205 manually picked spikes (Figure 3). Three-fold symmetry of the center-most spike was evident without applying any symmetry (Figure 3B) and was imposed in the subsequent rounds of refinement (Figure 3C). For detecting all the spikes automatically on the virion surfaces, we generated 106 seeds for each virion at the radius of 43 nm and spacing of 20 degrees (Figure 4A) and iteratively refined their positions relative to the membrane (Figure 4B).
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 
Figure 3: Refinement of the initial template structure. (A)&#160;Cylindrically averaged (C100) template constructed from manually defined positions of spikes. (B)&#160;Averaged density after five rounds of refinement without any symmetry (C1) imposed displays a spike with three-fold symmetric features. The resolution of the model is 48 &#197;. (C) Average of the spike was resolved at 41 &#197; after five rounds of refinement with three-fold symmetry.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 
Figure 4: Refinement of the seeds. A-B) A subset of seeds before (A) and after (B) refinement are shown on one virion density from Figure 2. Seeds in (B) have been color-coded based on the respective cross-correlation coefficients (blue, low correlation; red, high correlation).
The best correlating glycoprotein spike patches (top 75% after removing overlaps; ~1,000 spikes) were used to calculate the final average. The average was resolved to 35-&#197; resolution (Figure 5). It revealed a trimeric spike structure in the middle, in addition to some contribution from six neighboring spikes. Composite models of the virions, calculated by placing the structure in the known positions, revealed placement of the spikes on the virion surface (Figure 6A). Occasionally, locally ordered patches of spikes were evident (Figure 6B).
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 
Figure 5: Refined structure of a patch of the glycoprotein spike layer after template matching. (A-B) Two maps &#8216;even&#8217; and &#8216;odd&#8217;, reconstructed from two halves of the data are shown. The two maps show a remarkable degree of similarity, verifying the validness of the approach. The orientation around the spike long axis is different as the two maps have been reconstructed fully independently from each other. (C) Average of the two maps is shown at the final resolution of 35 &#197;.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 
Figure 6: Placement of the spikes on the Bunyamwera virion. (A) Composite model of a virion. View vectors (sticks) indicate the orientations of the spikes. Colors indicate the cross-correlation between each spike and the template structure (blue, low correlation; red, high correlation). (B) A close-up of an ordered patch of spikes.

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.2K Views.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Video: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Biochemistry

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. 
It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

Single-particle cryo-electron microscopy demands a suitable software package and user-friendly pipeline for high-throughput automatic data acquisition. Here, we present the application of a fully automated image acquisition software package, Latitude-S, and a practical pipeline for data collection of vitrified biomolecules under low-dose conditions.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Biology

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Samenvatting

Meer video's verkennen