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.).

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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 border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">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 ...

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Immunology and Infection

12.0K 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.