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- EMDB-3148: Electron cryo-microscopy of chikungunya virus in complex with neu... -

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Basic information

Entry
Database: EMDB / ID: EMD-3148
TitleElectron cryo-microscopy of chikungunya virus in complex with neutralizing antibody Fab 4J21
Map dataReconstruction of CHIKV VLP in complex with Fab molecules
Sample
  • Sample: Electron Cryo-microscopy of Chikungunya VLP in complex with human neutralizing antibody Fab 4J21
  • Virus: Chikungunya virus
  • Protein or peptide: Fab 4J21
Keywordschikungunya virus / neutralizing antibody Fab
Biological speciesHomo sapiens (human) / Chikungunya virus
Methodsingle particle reconstruction / cryo EM / Resolution: 16.5 Å
AuthorsLong F / Fong RH / Austin SK / Chen Z / Klose T / Fokine A / Liu Y / Porta J / Sapparapu G / Akahata W ...Long F / Fong RH / Austin SK / Chen Z / Klose T / Fokine A / Liu Y / Porta J / Sapparapu G / Akahata W / Doranz BJ / Crowe JE / Diamond MS / Rossmann MG
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity.
Authors: Feng Long / Rachel H Fong / Stephen K Austin / Zhenguo Chen / Thomas Klose / Andrei Fokine / Yue Liu / Jason Porta / Gopal Sapparapu / Wataru Akahata / Benjamin J Doranz / James E Crowe / ...Authors: Feng Long / Rachel H Fong / Stephen K Austin / Zhenguo Chen / Thomas Klose / Andrei Fokine / Yue Liu / Jason Porta / Gopal Sapparapu / Wataru Akahata / Benjamin J Doranz / James E Crowe / Michael S Diamond / Michael G Rossmann /
Abstract: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have ...Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain's β-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.
History
DepositionSep 8, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseNov 25, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3148.map.gz / Format: CCP4 / Size: 300.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of CHIKV VLP in complex with Fab molecules
Voxel sizeX=Y=Z: 2.39 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-15.54661655 - 15.48305511
Average (Standard dev.)0.09012548 (±1.98130405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-216-216-216
Dimensions432432432
Spacing432432432
CellA=B=C: 1032.4801 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.392.392.39
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z1032.4801032.4801032.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-216-216-216
NC/NR/NS432432432
D min/max/mean-15.54715.4830.090

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Supplemental data

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Sample components

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Entire : Electron Cryo-microscopy of Chikungunya VLP in complex with human...

EntireName: Electron Cryo-microscopy of Chikungunya VLP in complex with human neutralizing antibody Fab 4J21
Components
  • Sample: Electron Cryo-microscopy of Chikungunya VLP in complex with human neutralizing antibody Fab 4J21
  • Virus: Chikungunya virus
  • Protein or peptide: Fab 4J21

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Supramolecule #1000: Electron Cryo-microscopy of Chikungunya VLP in complex with human...

SupramoleculeName: Electron Cryo-microscopy of Chikungunya VLP in complex with human neutralizing antibody Fab 4J21
type: sample / ID: 1000
Oligomeric state: one Fab binds to one envelope protein on the surface of virus
Number unique components: 2

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Supramolecule #1: Chikungunya virus

SupramoleculeName: Chikungunya virus / type: virus / ID: 1 / Name.synonym: Chikungunya VLP / NCBI-ID: 37124 / Sci species name: Chikungunya virus / Sci species strain: strain 37997 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No / Syn species name: Chikungunya VLP
Host (natural)Organism: Aedes albopictus (Asian tiger mosquito) / synonym: INVERTEBRATES
Host systemOrganism: Homo sapiens (human) / Recombinant cell: FreeStyle 293-F
Virus shellShell ID: 1 / Diameter: 700 Å / T number (triangulation number): 4

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Macromolecule #1: Fab 4J21

MacromoleculeName: Fab 4J21 / type: protein_or_peptide / ID: 1 / Number of copies: 240 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: hybridoma
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa

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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

Concentration2 mg/mL
BufferpH: 7.2 / Details: PBS
GridDetails: 400 mesh ultrathin holey carbon copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 110 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 6 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60553 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJul 18, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 120 / Average electron dose: 22 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: JSPR / Number images used: 3149
DetailsThe particles were mannually selected using the program EMAN2.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: EMFit
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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