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- EMDB-25699: VFLIP Spike Trimer with GAR03 -

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Basic information

Entry
Database: EMDB / ID: EMD-25699
TitleVFLIP Spike Trimer with GAR03
Map data
Sample
  • Complex: VFLIP Spike trimer with GAR03 FAB
    • Protein or peptide: Spike glycoprotein
    • Protein or peptide: GAR03 Fab heavy chain
    • Protein or peptide: GAR03 Fab light chain
Keywordsspike / Fab / antibody / COVID / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesSevere acute respiratory syndrome-related coronavirus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsSobti M / Stewart AG
Funding support Australia, 1 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DE190100985 Australia
CitationJournal: Nat Commun / Year: 2023
Title: Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.
Authors: Romain Rouet / Jake Y Henry / Matt D Johansen / Meghna Sobti / Harikrishnan Balachandran / David B Langley / Gregory J Walker / Helen Lenthall / Jennifer Jackson / Stephanie Ubiparipovic / ...Authors: Romain Rouet / Jake Y Henry / Matt D Johansen / Meghna Sobti / Harikrishnan Balachandran / David B Langley / Gregory J Walker / Helen Lenthall / Jennifer Jackson / Stephanie Ubiparipovic / Ohan Mazigi / Peter Schofield / Deborah L Burnett / Simon H J Brown / Marianne Martinello / Bernard Hudson / Nicole Gilroy / Jeffrey J Post / Anthony Kelleher / Hans-Martin Jäck / Christopher C Goodnow / Stuart G Turville / William D Rawlinson / Rowena A Bull / Alastair G Stewart / Philip M Hansbro / Daniel Christ /
Abstract: Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies ...Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.
History
DepositionDec 12, 2021-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25699.png

Downloads & links

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Map

FileDownload / File: emd_25699.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-39.750731999999999 - 71.867670000000004
Average (Standard dev.)0.00022486754 (±1.020567)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : VFLIP Spike trimer with GAR03 FAB

EntireName: VFLIP Spike trimer with GAR03 FAB
Components
  • Complex: VFLIP Spike trimer with GAR03 FAB
    • Protein or peptide: Spike glycoprotein
    • Protein or peptide: GAR03 Fab heavy chain
    • Protein or peptide: GAR03 Fab light chain

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Supramolecule #1: VFLIP Spike trimer with GAR03 FAB

SupramoleculeName: VFLIP Spike trimer with GAR03 FAB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome-related coronavirus

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome-related coronavirus
Molecular weightTheoretical: 138.736516 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QCVNLTTRTQ LPPAYTNSFT RGVYYPDKVF RSSVLHSTQD LFLPFFSNVT WFHAIHVSGT NGTKRFDNPV LPFNDGVYFA STEKSNIIR GWIFGTTLDS KTQSLLIVNN ATNVVIKVCE FQFCNDPFLG VYYHKNNKSW MESEFRVYSS ANNCTFEYVS Q PFLMDLEG ...String:
QCVNLTTRTQ LPPAYTNSFT RGVYYPDKVF RSSVLHSTQD LFLPFFSNVT WFHAIHVSGT NGTKRFDNPV LPFNDGVYFA STEKSNIIR GWIFGTTLDS KTQSLLIVNN ATNVVIKVCE FQFCNDPFLG VYYHKNNKSW MESEFRVYSS ANNCTFEYVS Q PFLMDLEG KQGNFKNLRE FVFKNIDGYF KIYSKHTPIN LVRDLPQGFS ALEPLVDLPI GINITRFQTL LALHRSYLTP GD SSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NIT NLCPFG EVFNATRFAS VYAWNRKRIS NCVADYSVLY NSASFSTFKC YGVSPTKLND LCFTNVYADS FVIRGDEVRQ IAPG QTGKI ADYNYKLPDD FTGCVIAWNS NNLDSKVGGN YNYLYRLFRK SNLKPFERDI STEIYQAGST PCNGVEGFNC YFPLQ SYGF QPTNGVGYQP YRVVVLSFEL LHAPATVCGP KKSTNLVKNK CVNFNFNGLT GTGVLTESNK KFLPFQQFGR DIADTT DAV RDPQTLEILD ITPCSFGGVS VITPGTNTSN QVAVLYQGVN CTEVPVAIHA DQLTPTWRVY STGSNVFQTR AGCLIGA EH VNNSYECDIP IGAGICASYQ GGSGGSSIIA YTMSLGAENS VACSNNSIAI PTNFTISVTT EILPVSMTKT SVDCTMYI C GDSTECSNLL LQYGSFCTQL NRALTGIAVE QDKNTQEVFA QVKQIYKTPP IKDFGGFNFS QILPDPSKPS KRSPIEDLL FNKVTLADAG FIKQYGDCLG DIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI CSGWTFGAGP ALQIPFPMQM AYRFNGIGV TQNVLYENQK LIANQFNSAI GKIQDSLSST PSALGKLQDV VNQNAQALNT LVKQLSSNFG AISSVLNDIL S RLDKPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VF LHVTYVP AQEKNFTTAP AICHDGKAHF PREGVFVSNG THWFVTQRNF YEPQIITTDN TFVSGNCDVV IGIVNNTVYD PLQ PELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKGSGYIPE APRD GQAYV RKDGEWVLLS TFLLEVLFQG PAGWSHPQFE KGGGSGGGSG GGSWSHPQFE K

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Macromolecule #2: GAR03 Fab heavy chain

MacromoleculeName: GAR03 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.705656 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKKPGASVKV SCKASGYTFS SYDINWVRQA PGQGLEWMGW MSPNSGNTGY AQKFQGRVTM TRSTSMSTAY MELSSLRSE DTAVYFCARM SSSLTNYLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QVQLVQSGAE VKKPGASVKV SCKASGYTFS SYDINWVRQA PGQGLEWMGW MSPNSGNTGY AQKFQGRVTM TRSTSMSTAY MELSSLRSE DTAVYFCARM SSSLTNYLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCGSHHH HHH

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Macromolecule #3: GAR03 Fab light chain

MacromoleculeName: GAR03 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.970273 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SYVLTQPPSV AVAPGQTARI RCGENDIGSK NVHWYQQKSG QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEADYYCQV WDSTGDHPDV VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP ...String:
SYVLTQPPSV AVAPGQTARI RCGENDIGSK NVHWYQQKSG QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEADYYCQV WDSTGDHPDV VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTEC

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 130129
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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