+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13856 | ||||||||||||||||||
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Title | Tomogram of respiratory syncytial virus filamentous virion | ||||||||||||||||||
Map data | Representative tomogram of filamentous respiratory syncytial virus | ||||||||||||||||||
Sample |
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Keywords | Virion / VIRUS | ||||||||||||||||||
Biological species | Respiratory syncytial virus A2 | ||||||||||||||||||
Method | electron tomography / cryo EM | ||||||||||||||||||
Authors | Conley MJ / Vijayakrishnan S / Bhella D | ||||||||||||||||||
Funding support | United Kingdom, 5 items
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Citation | Journal: EMBO J / Year: 2022 Title: Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus. Authors: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia ...Authors: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia / Murray Stewart / Rachel Fearns / Swetha Vijayakrishnan / David Bhella / Abstract: Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image ...Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly. | ||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_13856.map.gz | 51.8 GB | EMDB map data format | |
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Header (meta data) | emd-13856-v30.xml emd-13856.xml | 12.8 KB 12.8 KB | Display Display | EMDB header |
Images | emd_13856.png | 184.5 KB | ||
Filedesc metadata | emd-13856.cif.gz | 5.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13856 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13856 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13856.map.gz / Format: CCP4 / Size: 56 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||
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Annotation | Representative tomogram of filamentous respiratory syncytial virus | ||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.79 Å | ||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : Respiratory syncytial virus A2
Entire | Name: Respiratory syncytial virus A2 |
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Components |
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-Supramolecule #1: Respiratory syncytial virus A2
Supramolecule | Name: Respiratory syncytial virus A2 / type: virus / ID: 1 / Parent: 0 Details: Sub tomogram averaging was performed focusing on the envelope of filamentous virions propagated directly on the cryo-EM grid. Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No |
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Host (natural) | Organism: Homo sapiens (human) |
-Supramolecule #2: Budding RSV Virion
Supramolecule | Name: Budding RSV Virion / type: organelle_or_cellular_component / ID: 2 / Parent: 1 |
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Source (natural) | Organism: Respiratory syncytial virus A2 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 |
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Grid | Model: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE Details: Grids were sterilised in EtOH and pretreated with laminin. |
Vitrification | Cryogen name: ETHANE |
Details | Virions were propagated in A549 cells, grown on gold quantifoil cryo-EM grids that had been pre-treated with laminin. |
Sectioning | Other: NO SECTIONING |
Fiducial marker | Manufacturer: BBI / Diameter: 5 nm |
-Electron microscopy
Microscope | TFS KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 2.0 e/Å2 Details: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered ...Details: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered direct detection camera. Dose-symmetric tilt-series collection was performed using SerialEM. Energy-filtered images were collected with a slit-width of 20eV and an applied defocus of between -2 and -4.5 microns. One second exposures were recorded with an electron exposure of 2 electrons/A2, partitioned over five movie frames. A total of 41 images were recorded per tilt-series at 3 degree intervals from -60 to +60, thus a total exposure of 82 electrons/A2 was applied per tilt-series. Tilt-series were recorded at a nominal column magnification of 81k corresponding to a calibrated pixel size of 1.79 A at the specimen scale. |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 41 |
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Details | Individual movies were corrected for drift and beam induced motion using MotionCor2. Motion corrected images were then compiled into an angle ordered stack file using a perl script to extract the correct files and tilt angles from the SerialEM metadata files (mdoc), creating the final tilt-series using the IMOD command new-stack. Tilt-series alignment and reconstruction by weighted back projection was then accomplished using the IMOD package |