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- EMDB-22751: SARS-CoV-2 Spike RBD in complex with neutralizing Fab 2H04 (local... -

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Basic information

Entry
Database: EMDB / ID: EMD-22751
TitleSARS-CoV-2 Spike RBD in complex with neutralizing Fab 2H04 (local refinement)
Map dataLocally refined map of SARS-CoV-2 Spike bound by 2H04 Fab
Sample
  • Complex: Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal antibody 2H04
    • Complex: SARS-CoV-2 Spike RBD
      • Protein or peptide: Spike protein S1
    • Complex: Fab fragment of monoclonal antibody 2H04
      • Protein or peptide: 2H04 heavy chain
      • Protein or peptide: 2H04 light chain
Function / homology
Function and homology information


Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell ...Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsErrico JM / Fremont DH / Center for Structural Genomics of Infectious Diseases (CSGID)
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)HHSN272201700060C United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93019C00062 United States
CitationJournal: Cell Rep / Year: 2021
Title: Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.
Authors: John M Errico / Haiyan Zhao / Rita E Chen / Zhuoming Liu / James Brett Case / Meisheng Ma / Aaron J Schmitz / Michael J Rau / James A J Fitzpatrick / Pei-Yong Shi / Michael S Diamond / Sean ...Authors: John M Errico / Haiyan Zhao / Rita E Chen / Zhuoming Liu / James Brett Case / Meisheng Ma / Aaron J Schmitz / Michael J Rau / James A J Fitzpatrick / Pei-Yong Shi / Michael S Diamond / Sean P J Whelan / Ali H Ellebedy / Daved H Fremont /
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron ...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.
History
DepositionSep 29, 2020-
Header (metadata) releaseSep 29, 2021-
Map releaseSep 29, 2021-
UpdateNov 10, 2021-
Current statusNov 10, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k9k
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7k9k
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22751.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally refined map of SARS-CoV-2 Spike bound by 2H04 Fab
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å
1.1 Å/pix.
x 300 pix.
= 330. Å
1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.5
Minimum - Maximum-2.5060174 - 4.808699
Average (Standard dev.)0.0023761163 (±0.070609584)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-2.5064.8090.002

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Supplemental data

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Mask #1

Fileemd_22751_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Truncated map used for model building and refinement

Fileemd_22751_additional_1.map
AnnotationTruncated map used for model building and refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map A

Fileemd_22751_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map B

Fileemd_22751_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal a...

EntireName: Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal antibody 2H04
Components
  • Complex: Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal antibody 2H04
    • Complex: SARS-CoV-2 Spike RBD
      • Protein or peptide: Spike protein S1
    • Complex: Fab fragment of monoclonal antibody 2H04
      • Protein or peptide: 2H04 heavy chain
      • Protein or peptide: 2H04 light chain

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Supramolecule #1: Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal a...

SupramoleculeName: Complex of SARS-CoV-2 Spike RBD with Fab fragment of monoclonal antibody 2H04
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragments generated by proteolytic cleavage of recombinantly expressed IgG

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Supramolecule #2: SARS-CoV-2 Spike RBD

SupramoleculeName: SARS-CoV-2 Spike RBD / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Supramolecule #3: Fab fragment of monoclonal antibody 2H04

SupramoleculeName: Fab fragment of monoclonal antibody 2H04 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Macromolecule #1: Spike protein S1

MacromoleculeName: Spike protein S1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 21.873496 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TNLCPFGEVF NATRFASVYA WNRKRISNCV ADYSVLYNSA SFSTFKCYGV SPTKLNDLCF TNVYADSFVI RGDEVRQIAP GQTGKIADY NYKLPDDFTG CVIAWNSNNL DSKVGGNYNY LYRLFRKSNL KPFERDISTE IYQAGSTPCN GVEGFNCYFP L QSYGFQPT ...String:
TNLCPFGEVF NATRFASVYA WNRKRISNCV ADYSVLYNSA SFSTFKCYGV SPTKLNDLCF TNVYADSFVI RGDEVRQIAP GQTGKIADY NYKLPDDFTG CVIAWNSNNL DSKVGGNYNY LYRLFRKSNL KPFERDISTE IYQAGSTPCN GVEGFNCYFP L QSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGP

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Macromolecule #2: 2H04 heavy chain

MacromoleculeName: 2H04 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.448784 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLQQSGAE LVKPGASVKM SCKASGYTFT SYWITWVKQR PGQGLEWIGD IYPGSGSTKY NEKFRSEATL TVDTSSTTAY MQLSSLTSE DSAVYYCARW DFYGSRTFDY WGQGTTLTVS SA

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Macromolecule #3: 2H04 light chain

MacromoleculeName: 2H04 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.460633 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
DIVLTQSPAI LSVSPGERVS FSCRASQNIG TIIHWYQQRT NGSPRLLIKY ASESVSGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQ QSSSWPLTFG AGTKLEL

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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 #1

Preparation ID1
Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
0.01 % w/vNaN3Sodium Azide
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 20s wait time 2s blot time.

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Sample preparation #2

Preparation ID2
Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
0.01 % w/vNaN3Sodium Azide
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 20s wait time 2s blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector.
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-45 / Average exposure time: 9.0 sec. / Average electron dose: 67.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 877481
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Details: C3 expanded particles / Number images used: 304667
FSC plot (resolution estimation)

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