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- EMDB-22499: Base-directed response of rabbit #1 week 12 polyclonal fragment a... -

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Basic information

Entry
Database: EMDB / ID: EMD-22499
TitleBase-directed response of rabbit #1 week 12 polyclonal fragment antigen binding in complex with 1PGE-THIVC SOSIP
Map datafinal map
Sample
  • Complex: Base-directed response of rabbit #1 week 12 polyclonal fragment antigen binding in complex with 1PGE-THIVC SOSIP
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / negative staining / Resolution: 29.0 Å
AuthorsSewell LM / Ozorowski G / Ward AB
Funding support United States, 4 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1084519 United States
Bill & Melinda Gates FoundationOPP1196345/INV-008813 United States
Bill & Melinda Gates FoundationOPP1115782 United States
Bill & Melinda Gates FoundationINV-002916 United States
CitationJournal: PLoS Pathog / Year: 2021
Title: Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer.
Authors: Rajesh Kumar / Suprit Deshpande / Leigh M Sewall / Gabriel Ozorowski / Christopher A Cottrell / Wen-Hsin Lee / Lauren G Holden / Sara T Richey / Antra Singh Chandrawacar / Kanika Dhiman / ...Authors: Rajesh Kumar / Suprit Deshpande / Leigh M Sewall / Gabriel Ozorowski / Christopher A Cottrell / Wen-Hsin Lee / Lauren G Holden / Sara T Richey / Antra Singh Chandrawacar / Kanika Dhiman / Ashish / Vivek Kumar / Shubbir Ahmed / Nitin Hingankar / Naresh Kumar / Kailapuri G Murugavel / Aylur K Srikrishnan / Devin Sok / Andrew B Ward / Jayanta Bhattacharya /
Abstract: Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational ...Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.
History
DepositionAug 20, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22499.png

Downloads & links

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Map

FileDownload / File: emd_22499.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.98 Å/pix.
x 192 pix.
= 380.16 Å		1.98 Å/pix.
x 192 pix.
= 380.16 Å		1.98 Å/pix.
x 192 pix.
= 380.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.98 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.045909543 - 0.11527015
Average (Standard dev.)0.00024781734 (±0.004892391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.981.981.98
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z380.160380.160380.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0460.1150.000

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Supplemental data

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Sample components

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Entire : Base-directed response of rabbit #1 week 12 polyclonal fragment a...

EntireName: Base-directed response of rabbit #1 week 12 polyclonal fragment antigen binding in complex with 1PGE-THIVC SOSIP
Components
  • Complex: Base-directed response of rabbit #1 week 12 polyclonal fragment antigen binding in complex with 1PGE-THIVC SOSIP

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Supramolecule #1: Base-directed response of rabbit #1 week 12 polyclonal fragment a...

SupramoleculeName: Base-directed response of rabbit #1 week 12 polyclonal fragment antigen binding in complex with 1PGE-THIVC SOSIP
type: complex / ID: 1 / Parent: 0
Details: IgG was purified from rabbit serum and digested into Fab using papain. Polyclonal fab was incubated with 1PGE-THIVC SOSIP trimers.
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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:
ConcentrationNameFormula
50.0 mMTris
150.0 mMSodium chlorideNaClSodium chloride
StainingType: NEGATIVE / Material: Uranyl formate / Details: 2% UF, stained once for 30-60 s prior to blotting
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: PARLODION / Support film - #0 - topology: CONTINUOUS / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING

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Electron microscopy

MicroscopeTFS TALOS F200C
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 73000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

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

4zmj

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 1734

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