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- EMDB-22999: Negative stain reconstruction of murine antibody M4H2K1 Fab in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-22999
TitleNegative stain reconstruction of murine antibody M4H2K1 Fab in complex with HIV-1 Env BG505 UFO.664 trimer
Map datafull map
Sample
  • Complex: 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 UFO.664
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / structural molecule activity / virion membrane / plasma membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsCopps J / Ozorowski G / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1UM1 AI144462 United States
CitationJournal: mBio / Year: 2021
Title: Neutralizing Antibodies Induced by First-Generation gp41-Stabilized HIV-1 Envelope Trimers and Nanoparticles.
Authors: Sonu Kumar / Xiaohe Lin / Timothy Ngo / Benjamin Shapero / Cindy Sou / Joel D Allen / Jeffrey Copps / Lei Zhang / Gabriel Ozorowski / Linling He / Max Crispin / Andrew B Ward / Ian A Wilson / Jiang Zhu /
Abstract: The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and ...The immunogenicity of gp41-stabilized HIV-1 BG505 envelope (Env) trimers and nanoparticles (NPs) was recently assessed in mice and rabbits. Here, we combined Env-specific B-cell sorting and repertoire sequencing to identify neutralizing antibodies (NAbs) from immunized animals. A panel of mouse NAbs was isolated from mice immunized with a 60-meric I3-01 NP presenting 20 stabilized trimers. Three mouse NAbs potently neutralized BG505.T332N by recognizing a glycan epitope centered in the C3/V4 region on BG505 Env, as revealed by electron microscopy (EM), X-ray crystallography, and epitope mapping. A set of rabbit NAbs was isolated from rabbits immunized with a soluble trimer and a 24-meric ferritin NP presenting 8 trimers. Neutralization assays against BG505.T332N variants confirmed that potent rabbit NAbs targeted previously described glycan holes on BG505 Env and accounted for a significant portion of the autologous NAb response in both the trimer and ferritin NP groups. Last, we examined NAb responses that were induced by non-BG505 Env immunogens. We determined a 3.4-Å-resolution crystal structure for the clade C transmitted/founder (T/F) Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend in gp41. This clade C Env, in a soluble trimer form and in a multivalent form with 8 trimers attached to ferritin NP, and the gp41-stabilized clade A Q482-d12 Env trimer elicited distinct NAb responses in rabbits, with notable differences in neutralization breadth. Although eliciting a broad NAb response remains a major challenge, our study provides valuable information on an HIV-1 vaccine design strategy that combines gp41 stabilization and NP display. Self-assembling protein nanoparticles (NPs) presenting BG505 envelope (Env) trimers can elicit tier 2 HIV-1-neutralizing antibody (NAb) responses more effectively than soluble trimers. In the present study, monoclonal NAbs were isolated from previously immunized mice and rabbits for structural and functional analyses, which revealed that potent mouse NAbs recognize the C3/V4 region and small NP-elicited rabbit NAbs primarily target known glycan holes on BG505 Env. This study validates the gp41 stabilization strategy for HIV-1 Env vaccine design and highlights the challenge in eliciting a broad NAb response.
History
DepositionNov 17, 2020-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22999.png

Downloads & links

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Map

FileDownload / File: emd_22999.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-0.6475193 - 3.8630776
Average (Standard dev.)0.062333666 (±0.36063755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.6483.8630.062

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Supplemental data

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Sample components

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Entire : 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 U...

EntireName: 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 UFO.664
Components
  • Complex: 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 UFO.664

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Supramolecule #1: 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 U...

SupramoleculeName: 3 copies of M4H2K1 Fab in complex with trimeric HIV-1 Env BG505 UFO.664
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F
Molecular weightTheoretical: 570 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
50.0 mMTris
150.0 mMSodium chloride
StainingType: NEGATIVE / Material: uranyl formate / Details: 2% (w/v) UF applied for 60 s
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zmj


Details: Map created from PDB coordinates and low pass filtered
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. v2) / Number images used: 14027
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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