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- EMDB-23779: BG505 SOSIP.v5.2 in complex with the polyclonal Fab pAbC-1 from a... -

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Basic information

Entry
Database: EMDB / ID: EMD-23779
TitleBG505 SOSIP.v5.2 in complex with the polyclonal Fab pAbC-1 from animal Rh.4O9
Map data3D map of BG505 SOSIP v5.2 in complex with the polyclonal Fab pAbC-1 from animal Rh.4O9. Main map
Sample
  • Complex: BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolated from animal Rh4O9.
    • Protein or peptide: Rh4O9.8 monoclonal antibody Light Chain
    • Protein or peptide: Rh4O9.8 monoclonal antibody Heavy Chain
  • Protein or peptide: Surface protein gp120
  • Protein or peptide: Transmembrane protein gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / virion membrane / structural molecule activity / identical protein binding / 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 speciesHuman immunodeficiency virus / Human immunodeficiency virus 1 / Macaca mulatta (Rhesus monkey)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsAntanasijevic A / Ozorowski G / Nogal B / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
Bill & Melinda Gates FoundationOPP1196345 United States
CitationJournal: Sci Adv / Year: 2022
Title: From structure to sequence: Antibody discovery using cryoEM.
Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A ...Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A Enemuo / Leigh M Sewall / Bartek Nogal / Fangzhu Zhao / Bettina Groschel / William R Schief / Devin Sok / Guido Silvestri / Shane Crotty / Steven E Bosinger / Andrew B Ward /
Abstract: One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and ...One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.
History
DepositionApr 6, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mdt
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mdt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23779.png

Downloads & links

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Map

FileDownload / File: emd_23779.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of BG505 SOSIP v5.2 in complex with the polyclonal Fab pAbC-1 from animal Rh.4O9. Main map
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.025
Minimum - Maximum-0.066957034 - 0.124311276
Average (Standard dev.)-0.00015122425 (±0.0033262707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 370.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z370.800370.800370.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0670.124-0.000

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Supplemental data

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Mask #1

Fileemd_23779_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3D map of BG505 SOSIP v5.2 in complex...

Fileemd_23779_half_map_1.map
Annotation3D map of BG505 SOSIP v5.2 in complex with the polyclonal Fab pAbC-1 from animal Rh.4O9. Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3D map of BG505 SOSIP v5.2 in complex...

Fileemd_23779_half_map_2.map
Annotation3D map of BG505 SOSIP v5.2 in complex with the polyclonal Fab pAbC-1 from animal Rh.4O9. Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolat...

EntireName: BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolated from animal Rh4O9.
Components
  • Complex: BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolated from animal Rh4O9.
    • Protein or peptide: Rh4O9.8 monoclonal antibody Light Chain
    • Protein or peptide: Rh4O9.8 monoclonal antibody Heavy Chain
  • Protein or peptide: Surface protein gp120
  • Protein or peptide: Transmembrane protein gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolat...

SupramoleculeName: BG505 SOSIP.v5.2 in complex with the polyclonal Fab sample isolated from animal Rh4O9.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3-#4
Details: The map was generated from polyclonal Fab sample isolated from animal Rh4O9. The polyclonal Fabs were complexed with BG505 SOSIP.v5.2 and imaged. Rh4O9.8 monoclonal antibody sequence was ...Details: The map was generated from polyclonal Fab sample isolated from animal Rh4O9. The polyclonal Fabs were complexed with BG505 SOSIP.v5.2 and imaged. Rh4O9.8 monoclonal antibody sequence was relaxed into the Fab-corresponding density.
Source (natural)Organism: Human immunodeficiency virus
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F / Recombinant plasmid: pPPI4

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Macromolecule #1: Surface protein gp120

MacromoleculeName: Surface protein gp120 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 57.775883 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS LFYRLDVVQI NENQGNRSNN SNKEYRLINC NTSAITQACP KVSFEPIPIH YCAPAGFAIL KCKDKKFNGT GP CPSVSTV QCTHGIKPVV STQLLLNGSL AEEEVMIRSE NITNNAKNIL VQFNTPVQIN CTRPNNNTRK SIRIGPGQWF YAT GDIIGD IRQAHCNVSK ATWNETLGKV VKQLRKHFGN NTIIRFANSS GGDLEVTTHS FNCGGEFFYC NTSGLFNSTW ISNT SVQGS NSTGSNDSIT LPCRIKQIIN MWQRIGQAMY APPIQGVIRC VSNITGLILT RDGGSTNSTT ETFRPGGGDM RDNWR SELY KYKVVKIEPL GVAPTRCKRR VVGRRRRRR

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Macromolecule #2: Transmembrane protein gp41

MacromoleculeName: Transmembrane protein gp41 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 17.178549 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AVGIGAVFLG FLGAAGSTMG AASMTLTVQA RNLLSGIVQQ QSNLLRAPEC QQHLLKLTVW GIKQLQARVL AVERYLRDQQ LLGIWGCSG KLICCTNVPW NSSWSNRNLS EIWDNMTWLQ WDKEISNYTQ IIYGLLEESQ NQQEKNEQDL LALD

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Macromolecule #3: Rh4O9.8 monoclonal antibody Light Chain

MacromoleculeName: Rh4O9.8 monoclonal antibody Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 11.339304 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QAGLTQPPSA SEAAGKSVTI SCSGSSANIG STSVSWYQQL PGTAPKLLIY YNDQRASGVS DRFSGSKSGT SASLAISGLQ TEDEADYYC AAWDDNLSGR IFGGGTRLTV L

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Macromolecule #4: Rh4O9.8 monoclonal antibody Heavy Chain

MacromoleculeName: Rh4O9.8 monoclonal antibody Heavy Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 12.736094 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLQESGGG LAQPGGSLRL TCEASGFTFG RDDMAWVRQA LGKGLEWVSS ISNSGNTIYY ADPVKGRFSI SRDNAKNSLS LQMNSLKIE DTAVYFCTRT LGDYYLDWGQ GVQVTVSS

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 31 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration1.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMTris-HClTris-HCl
150.0 mMNaClSodium chloride

Details: TBS, 0.2um filtered
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
DetailsThe complex was generated by incubation of Rh4O9 polyclonal antibody (as Fab) with recombinantly expressed BG505 SOSIP.v5.2 and subsequent SEC purification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 13.5 sec. / Average electron dose: 61.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 327121 / Details: Template picker in cryoSPARC
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Low-pass-filtered map of BG505 SOSIP
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 98122
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Relion, regularized likelihood
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Relion, regularized likelihood
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5cez

Output model

PDB-7mdt:
BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)

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