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- EMDB-20346: Polyclonal serum of rabbit A1 post 6th immunization in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-20346
TitlePolyclonal serum of rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5 NFL TD 2CC+, class 1
Map dataPolyclonal Fabs from rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5
Sample
  • Complex: Polyclonal serum of rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5 NFL TD 2CC+, class 1
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsTurner HL / Andrew AB
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesUM AI100663 United States
Bill & Melinda Gates FoundationOPP1084519 United States
Bill & Melinda Gates FoundationOPP1115782 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI136621 United States
CitationJournal: Immunity / Year: 2019
Title: Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.
Authors: Viktoriya Dubrovskaya / Karen Tran / Gabriel Ozorowski / Javier Guenaga / Richard Wilson / Shridhar Bale / Christopher A Cottrell / Hannah L Turner / Gemma Seabright / Sijy O'Dell / Jonathan ...Authors: Viktoriya Dubrovskaya / Karen Tran / Gabriel Ozorowski / Javier Guenaga / Richard Wilson / Shridhar Bale / Christopher A Cottrell / Hannah L Turner / Gemma Seabright / Sijy O'Dell / Jonathan L Torres / Lifei Yang / Yu Feng / Daniel P Leaman / Néstor Vázquez Bernat / Tyler Liban / Mark Louder / Krisha McKee / Robert T Bailer / Arlette Movsesyan / Nicole A Doria-Rose / Marie Pancera / Gunilla B Karlsson Hedestam / Michael B Zwick / Max Crispin / John R Mascola / Andrew B Ward / Richard T Wyatt /
Abstract: The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are ...The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.
History
DepositionJun 25, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseNov 20, 2019-
UpdateApr 1, 2020-
Current statusApr 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0129
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0129
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20346.png

Downloads & links

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Map

FileDownload / File: emd_20346.map.gz / Format: CCP4 / Size: 20.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPolyclonal Fabs from rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.79 Å/pix.
x 176 pix.
= 315.04 Å		1.79 Å/pix.
x 176 pix.
= 315.04 Å		1.79 Å/pix.
x 176 pix.
= 315.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.79 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0229 / Movie #1: 0.0129
Minimum - Maximum-0.038345218 - 0.07806933
Average (Standard dev.)0.00011349281 (±0.0044259457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 315.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.791.791.79
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z315.040315.040315.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.0380.0780.000

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Supplemental data

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Sample components

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Entire : Polyclonal serum of rabbit A1 post 6th immunization in complex wi...

EntireName: Polyclonal serum of rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5 NFL TD 2CC+, class 1
Components
  • Complex: Polyclonal serum of rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5 NFL TD 2CC+, class 1

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Supramolecule #1: Polyclonal serum of rabbit A1 post 6th immunization in complex wi...

SupramoleculeName: Polyclonal serum of rabbit A1 post 6th immunization in complex with heterologous Env trimer 45_01dG5 NFL TD 2CC+, class 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
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

Concentration0.03 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
50.0 mMTris
150.0 mMSodium chloride
StainingType: NEGATIVE / Material: uranyl formate
Details: Grids were negatively stained with 2% (w/v) uranyl formate for 50 s

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: OTHER / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zmj


Details: Coordinates for ligand-free SOSIP trimer converted to EM map and low-pass filtered to 40 Angstrom
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 6745
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)

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