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- EMDB-9340: Cryo-EM map of VEEV-TC-83 fixed with 1% glutaraldehyde -

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Basic information

Entry
Database: EMDB / ID: EMD-9340
TitleCryo-EM map of VEEV-TC-83 fixed with 1% glutaraldehyde
Map data3D map of vaccine strain of venezuelan equine encephalitis virus (TC-83) inactivated by fixing with 1% glutaraldehyde
Sample
  • Virus: Venezuelan equine encephalitis virus
Biological speciesVenezuelan equine encephalitis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 7.87 Å
AuthorsParvate AD / Jonsson CJ
CitationJournal: J Virol Methods / Year: 2020
Title: A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV).
Authors: Amar D Parvate / Frank Vago / S Saif Hasan / Jasper Lee / Evan P Williams / Jason Lanman / Colleen B Jonsson /
Abstract: The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address ...The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.
History
DepositionNov 14, 2018-
Header (metadata) releaseJan 16, 2019-
Map releaseNov 20, 2019-
UpdateJan 8, 2020-
Current statusJan 8, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.87
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.87
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9340.png

Downloads & links

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Map

FileDownload / File: emd_9340.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of vaccine strain of venezuelan equine encephalitis virus (TC-83) inactivated by fixing with 1% glutaraldehyde
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.62 Å/pix.
x 560 pix.
= 906.08 Å		1.62 Å/pix.
x 560 pix.
= 906.08 Å		1.62 Å/pix.
x 560 pix.
= 906.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.618 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.87 / Movie #1: 1.87
Minimum - Maximum-11.705414 - 14.805672
Average (Standard dev.)0.00000000000 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-280-280-280
Dimensions560560560
Spacing560560560
CellA=B=C: 906.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.6181.6181.618
M x/y/z560560560
origin x/y/z0.0000.0000.000
length x/y/z906.080906.080906.080
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS-280-280-280
NC/NR/NS560560560
D min/max/mean-11.70514.8060.000

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Supplemental data

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Sample components

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Entire : Venezuelan equine encephalitis virus

EntireName: Venezuelan equine encephalitis virus
Components
  • Virus: Venezuelan equine encephalitis virus

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Supramolecule #1: Venezuelan equine encephalitis virus

SupramoleculeName: Venezuelan equine encephalitis virus / type: virus / ID: 1 / Parent: 0
Details: TC-83 was initially reported in 1961 after 45 serial passages in guinea pig heart cells
NCBI-ID: 11036 / Sci species name: Venezuelan equine encephalitis virus / Sci species strain: TC-83 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Virus shellShell ID: 1 / Diameter: 690.0 Å / T number (triangulation number): 4

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2 / Details: Tris 0.01M, NaCl 0.1M, EDTA 0.001M
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 6 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 483 / Average exposure time: 11.0 sec. / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: -3.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: -1.0 µm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 12909
CTF correctionSoftware - Name: Gctf
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: jspr / Number images used: 12849
Initial angle assignmentType: OTHER / Software - Name: jspr
Final angle assignmentType: OTHER
Final 3D classificationSoftware - Name: RELION (ver. 2.1)

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