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- EMDB-29492: Three-Dimensional Reconstruction of HCV Envelope Glycoproteins E1... -

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Entry
Database: EMDB / ID: EMD-29492
TitleThree-Dimensional Reconstruction of HCV Envelope Glycoproteins E1E2 Heterodimer by Electron Microscopic Analysis
Map data
Sample
  • Complex: HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex
Biological speciesHepacivirus C
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsKanai T / Liang TJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: J Virol / Year: 2023
Title: Three-Dimensional Reconstruction of the Hepatitis C Virus Envelope Glycoprotein E1E2 Heterodimer by Electron Microscopic Analysis.
Authors: Tapan Kanai / Zongyi Hu / Renbin Yang / Weimin Wu / Ziqiu Wang / Christopher D Ma / Jaime Sanchez-Meza / Mansun Law / Michael Houghton / John Lokman Law / Michael Logan / Natalia de Val / T Jake Liang /
Abstract: Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, ...Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here. Recombinant HCV E1E2 heterodimer is being developed as a vaccine candidate. Using electron microscopy, we demonstrated unique features of E1E2 in complex with various neutralizing antibodies and small molecule inhibitors that are important to understanding its antigenicity and induction of immune response.
History
DepositionJan 19, 2023-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateFeb 15, 2023-
Current statusFeb 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29492.png

Downloads & links

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Map

FileDownload / File: emd_29492.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.033231534 - 0.06614537
Average (Standard dev.)5.1981853e-05 (±0.0009970411)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29492_half_map_1.map
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Half map: #2

Fileemd_29492_half_map_2.map
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Sample components

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Entire : HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex

EntireName: HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex
Components
  • Complex: HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex

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Supramolecule #1: HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex

SupramoleculeName: HCV Envelope Glycoproteins E1E2 Heterodimer-Fab complex
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Hepacivirus C

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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
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: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.0 µm / Nominal defocus min: -2.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 167000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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