Name: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo (Video) | JoVE Journal
Uploaded: 2014-10-21T13:00:00.000+00:00
Duration: 8 min 29 s
Description: Learn to average viral envelope glycoprotein spikes using electron cryo-tomography and Jsubtomo software. This computational method resolves trimeric structures at 35 angstrom resolution for studying viral pathogenesis and drug design.

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5 minutes

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

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 (Å)</td>
			<td>5.4</td>
		</tr>
		<tr>
			<td>Estimated dose (e–/ Å2)</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Underfocus (μm)</td>
			<td>4.0–4.5</td>
		</tr>
		<tr>
			<td>First CTF zero (Å)a</td>
			<td>26–30</td>
		</tr>
		<tr>
			<td>Tilt range (°)</td>
			<td>–60–60</td>
		</tr>
		<tr>
			<td>Tilt sampling (°)</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 (°)</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Resolution range used in the refinement (Å)</td>
			<td>42–334</td>
		</tr>
		<tr>
			<td>Final resolution estimate (Å)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="Virus-like particle structure; cryo-electron microscopy image; nanostructure analysis." fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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 Å. 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="3D molecular surface models, top and side view diagram, structural analysis, computational study." fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3: Refinement of the initial template structure. (A) Cylindrically averaged (C100) template constructed from manually defined positions of spikes. (B) 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 Å. (C) Average of the spike was resolved at 41 Å after five rounds of refinement with three-fold symmetry.
<img alt="Circular diagram with vectors indicating direction, suggesting molecular or structural analysis." fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
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-Å 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="3D surface models of protein structure; top and side views showing even, odd, average configurations." fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5: Refined structure of a patch of the glycoprotein spike layer after template matching. (A-B) Two maps ‘even’ and ‘odd’, 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 Å.
<img alt="Protein structure model (A) and density map (B) showing molecular surface visualization." fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
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.

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 ...

The overall goal of this procedure is to study the structures of glycoprotein complexes present on the surfaces of enveloped viruses. This is accomplished by first locating the virus particles in the tomographic volumes that have been previously calculated from electron cryo microscopy data. The second step is to create two statistically independent initial models of the glycoprotein complexes using a manually defined subset of data.Next, these models are refined and then used to automatically detect all glycoprotein complexes on the virus particles. The final step is to refine the structure of the glycoprotein complex using the full data set. Ultimately, molecular visualization tools are used to interpret the final structure.Three dimensional structural analysis of these glycoprotein spikes is invaluable for understanding viral pathogenesis as well as in drug design. The main advantage of this technique over existing methods, such as x-ray crystallography, is that this technique allows solving structures of membrane proteins in the natural membrane environment. This technique can help to address key topics in structural biology.For example, the structure of many membrane containing viruses. Although this technique can provide insight into glycoprotein complexes in these viruses, it can also be applied to other systems such as membrane proteins incorporated into liposomes. The procedure will be demonstrated by two members of my group, Slee and David Bittel.To begin open a autogram file in B show. Pick virus particles manually in the grams by defining the center coordinate of a virus particle using the particle picking tool. Repeat this step until all variants have been processed.Save virus coordinates in a star file. Proceed to repeat this process for all tomos. Next run J sub tomo py in extract mode To extract vari on sub volumes into individual volume files, use the saved star files as input files to generate two independent initial models.First, open ovarian sub volume map file. In B show. Pick a subset of spikes in the Varian sub volumes by defining the center coordinate of a spike using the particle picking tool accessible via the toolbox window.Repeat this step until all clearly distinct spikes have been processed. Save spike coordinates in a star file. Repeat this step until approximately 200 glycoprotein spikes have been processed.After assigning views to the varians as described in the text protocol, create a real space mask using J sub tomo. Create mask py. Then generate a reciprocal space mask using J sub tomo create wedge mask py.The reciprocal space mask is used to exclude regions in the missing wedge region, resulting from single axis tomographic data collection. Next, create two initial averages using J.Sub tomo create averages. PY using a saved selection file as the input file.Use ization to reduce noise in the initial averages is iteratively align and average the two previously generated models with J sub tomo iterate gold PY.Details for both stages of this process can be found in the text protocol. Assess the degree of symmetry in the structure by examining the resulting low-pass filtered map file in kyira. For example, if the spike is a trimeric complex, threefold symmetry should be evident.Continue refinement of the structure as detailed in the text protocol. Next, generate seeds located evenly on the Varian surface for template matching and assign an initial view vector to the seeds by running JVs py. Use the previously generated star files as input files generate approximately 1.5 times more seeds than the expected number of spikes.This view vector approximates the direction of the spike closest to each seed point. Next, generate two independent averages of the virus surface as before. Then refine the position of the seeds as detailed in the text protocol.Finally, generate com marker files of the refined seed star files using jvs.py. Examine the seeds by opening the CMM files and the associated virion map files. In kyira.Make sure that the refined seeds are aligned correctly relative to the virus membrane. The refined markers can be colored based on their cross correlation Coefficients automatically locate all the spikes in the Varian sub volumes using local template matching around refined seeds align and average the located spikes use the averages generated from a subset of manually picked spikes as initial templates. Additional details for this process can be found in the text protocol to visualize the results, open the refined structure of the spike in kyira for visualization.Proceed to fit the atomic structures. Then create a composite model of the virion using j Subo Create model DO py. Finally open the composite model for visualization in kyira.The initial model was refined using 205 manually picked spikes.Threefold. Symmetry of the centermost spike was evident without applying any symmetry and was imposed on the subsequent rounds of refinement for detecting all the spikes manually on the Varian surfaces. 106 seeds were generated for each vion at the radius of 43 nanometers and spacing of 20 degrees, and their positions were iteratively refined relative to the membrane.The best correlating glycoprotein spike patches were used to calculate the final average. The average was resolved to 35 angstrom resolution. It revealed a trimeric spike structure in the middle in addition to some contribution from six neighboring spikes.Composite models of the varis calculated by placing the structure in the known positions revealed placement of spikes on the Varian surface. Occasionally, locally ordered patches of spikes were evident. While attempting this procedure, it's important to use only the best data and to carefully check all the intermediate results.Following this procedure, x-ray crystallography structures can be fitted into the final average to get a more accurate view of protein-protein interactions. After watching this video, you should have a good understanding of how to average viral envelope glycoprotein structures using J Omo.

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Research

JoVE Journal

Immunology and Infection

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...

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

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.

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

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.

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

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.

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

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.

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

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

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

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 Å resolution.

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

This video protocol illustrates how to perform scanning transmission electron microscopy tomography of virological specimens. For optimal outcomes, samples are prepared by high-pressure freezing and subsequent freeze substitution.

Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples

This article presents a protocol for data processing of influenza viruses imaged using cryo-electron tomography and subsequent subtomogram averaging of the hemagglutinin glycoprotein. This protocol covers step-by-step data processing, from image preprocessing to final model refinement.

Subnanometer-Resolution Structural Determination of Hemagglutinin from Cryo-Electron Tomography of Influenza Viruses

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology.  Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Learn to average viral envelope glycoprotein spikes using electron cryo-tomography and Jsubtomo software. This computational method resolves trimeric structures at 35 angstrom resolution for studying viral pathogenesis and drug design.

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

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