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- EMDB-9328: Pre-fusion protein gB revealed on human cytomegalovirus by cryo e... -

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Basic information

Entry
Database: EMDB / ID: EMD-9328
TitlePre-fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate
Map dataIn situ structure of HCMV gB in the prefusion conformation
Sample
  • Virus: Human herpesvirus 5 strain AD169
Biological speciesHuman herpesvirus 5 strain AD169
Methodsubtomogram averaging / cryo EM / Resolution: 21.6 Å
AuthorsZhu S / Jiayan Z
CitationJournal: PLoS Pathog / Year: 2018
Title: Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.
Authors: Zhu Si / Jiayan Zhang / Sakar Shivakoti / Ivo Atanasov / Chang-Lu Tao / Wong H Hui / Kang Zhou / Xuekui Yu / Weike Li / Ming Luo / Guo-Qiang Bi / Z Hong Zhou /
Abstract: Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although ...Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain.
History
DepositionNov 12, 2018-
Header (metadata) releaseNov 28, 2018-
Map releaseNov 28, 2018-
UpdateDec 19, 2018-
Current statusDec 19, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.53
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.53
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9328.png

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Map

FileDownload / File: emd_9328.map.gz / Format: CCP4 / Size: 549.8 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure of HCMV gB in the prefusion conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.2 Å/pix.
x 52 pix.
= 270.4 Å		5.2 Å/pix.
x 52 pix.
= 270.4 Å		5.2 Å/pix.
x 52 pix.
= 270.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.53 / Movie #1: 1.53
Minimum - Maximum-3.5451634 - 5.5776234
Average (Standard dev.)0.0000011746909 (±1.)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-26-26-26
Dimensions525252
Spacing525252
CellA=B=C: 270.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.25.25.2
M x/y/z525252
origin x/y/z0.0000.0000.000
length x/y/z270.400270.400270.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-26-26-26
NC/NR/NS525252
D min/max/mean-3.5455.5780.000

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Supplemental data

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Sample components

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Entire : Human herpesvirus 5 strain AD169

EntireName: Human herpesvirus 5 strain AD169
Components
  • Virus: Human herpesvirus 5 strain AD169

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Supramolecule #1: Human herpesvirus 5 strain AD169

SupramoleculeName: Human herpesvirus 5 strain AD169 / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 10360 / Sci species name: Human herpesvirus 5 strain AD169 / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: Yes
Molecular weightTheoretical: 300 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 21.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 39
ExtractionNumber tomograms: 39 / Number images used: 1509
Final angle assignmentType: NOT APPLICABLE

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