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- PDB-8cwk: Fab arm of antibodies 4G1-C2 and 10G4 bound to CoV-2 receptor bin... -

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Basic information

Entry
Database: PDB / ID: 8cwk
TitleFab arm of antibodies 4G1-C2 and 10G4 bound to CoV-2 receptor binding domain (RBD)
Components
  • (Heavy chain of Fab arm of antibody ...) x 2
  • (Light chain of Fab arm of antibody ...) x 2
  • Spike protein S1
KeywordsIMMUNE SYSTEM / antibody / CoV-2 / receptor binding domain / class 5 epitope
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / membrane fusion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / extracellular region / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set ...: / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Spike glycoprotein / Ig-like domain-containing protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus 2
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.368 Å
AuthorsLangley, D.B. / Christ, D.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia) Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Affinity maturation endows potent activity onto class 6 SARS-CoV-2 broadly neutralizing antibodies.
Authors: Ohan Mazigi / David B Langley / Jake Y Henry / Deborah L Burnett / Meghna Sobti / Gregory J Walker / Romain Rouet / Harikrishnan Balachandran / Helen Lenthall / Jennifer Jackson / Stephanie ...Authors: Ohan Mazigi / David B Langley / Jake Y Henry / Deborah L Burnett / Meghna Sobti / Gregory J Walker / Romain Rouet / Harikrishnan Balachandran / Helen Lenthall / Jennifer Jackson / Stephanie Ubiparipovic / Peter Schofield / Simon H J Brown / Sebastian R Schulz / Markus Hoffmann / Stefan Pöhlmann / Jeffrey Post / Marianne Martinello / Golo Ahlenstiel / Anthony Kelleher / William D Rawlinson / Stuart G Turville / Rowena A Bull / Alastair G Stewart / Hans-Martin Jäck / Christopher C Goodnow / Daniel Christ /
Abstract: The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails ...The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails and of first-generation broadly neutralizing antibodies such as S309 (Sotrovimab). In contrast, antibodies targeting cryptic conformational epitopes of the receptor binding domain (RBD) have demonstrated broad activity against emerging variants, but exert only moderate neutralizing activity, which has so far hindered clinical development. Here, we utilize in vitro display technology to identify and affinity-mature antibodies targeting the cryptic class 6 epitope, accessible only in the "up" conformation of the SARS-CoV-2 spike trimer. Increasing antibody affinity into the low picomolar range endowed potent neutralization of VOCs and protection of hACE2 mice from viral challenge. Cryoelectron microscopy and crystal structures of two affinity-matured antibodies (4C12-B12 and 4G1-C2) in complex with RBD highlighted binding modes and epitopes distal from mutational hotspots commonly overserved in VOCs, providing direct structural insights into the observed mutational resistance. Moreover, we further demonstrate that antibodies targeting the class 6 epitope, rather than being an artifact of in vitro selection, are common in the IgG1 memory B cell repertoire of convalescent patients and can be induced in human antibody V-gene transgenic mice through immunization. Our results highlight the importance of very high (picomolar) affinity in the development of neutralizing antibodies and vaccines and suggest an affinity threshold in the provision of broad and long-lasting immunity against SARS-CoV-2.
History
DepositionMay 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification
Revision 1.3Jan 15, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Heavy chain of Fab arm of antibody 10G4
L: Light chain of Fab arm of antibody 10G4
A: Heavy chain of Fab arm of antibody 4G1-C2
B: Light chain of Fab arm of antibody 4G1-C2
C: Spike protein S1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,46113
Polymers119,4665
Non-polymers9958
Water3,189177
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12080 Å2
ΔGint-56 kcal/mol
Surface area45050 Å2
MethodPISA
Unit cell
Length a, b, c (Å)133.155, 99.343, 124.957
Angle α, β, γ (deg.)90.000, 108.630, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Antibody , 4 types, 4 molecules HLAB

#1: Antibody Heavy chain of Fab arm of antibody 10G4


Mass: 24475.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Light chain of Fab arm of antibody 10G4


Mass: 23591.232 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Heavy chain of Fab arm of antibody 4G1-C2


Mass: 24809.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody Light chain of Fab arm of antibody 4G1-C2


Mass: 23614.150 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: Q6GMX0

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Protein / Sugars , 2 types, 3 molecules C

#5: Protein Spike protein S1


Mass: 22975.688 Da / Num. of mol.: 1 / Fragment: receptor binding domain (RBD)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P0DTC2
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 183 molecules

#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 177 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.27 Å3/Da / Density % sol: 62.42 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: Equal volume (2 uL) of protein solution (approx 5 mg/mL, in 25 mM Tris (pH 8.0), 200 mM NaCl) was mixed with an equal volume of well solution comprising 200 mM sodium citrate, 100 mM Bis- ...Details: Equal volume (2 uL) of protein solution (approx 5 mg/mL, in 25 mM Tris (pH 8.0), 200 mM NaCl) was mixed with an equal volume of well solution comprising 200 mM sodium citrate, 100 mM Bis-Tris-Propane (pH 7.4), 18% PEG3350). For cryoprotection the crystal was swum briefly (5-10 sec) in well solution doped with glycerol to a final concentration of ~25%.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9536 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 21, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9536 Å / Relative weight: 1
ReflectionResolution: 2.368→45.8 Å / Num. obs: 62334 / % possible obs: 99.3 % / Redundancy: 5.9 % / Biso Wilson estimate: 53.77 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.092 / Rpim(I) all: 0.041 / Rrim(I) all: 0.101 / Net I/σ(I): 10.9
Reflection shell

Diffraction-ID: 1 / Redundancy: 5.8 %

Resolution (Å)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.37-2.431.0292543043930.6980.4551.1271.595.3
10.59-45.80.05242147320.9970.0230.05829.198.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.24 Å45.8 Å
Translation3.24 Å45.8 Å

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Processing

Software
NameVersionClassification
PHENIX1.11.1_2575refinement
XDSdata reduction
Aimless0.7.4data scaling
PHASER2.8.3phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: generic Fab and RBD

Resolution: 2.368→43.235 Å / SU ML: 0.33 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 25.98 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2232 3116 5 %
Rwork0.1863 59197 -
obs0.1882 62313 99.16 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 139.78 Å2 / Biso mean: 58.7074 Å2 / Biso min: 29.59 Å2
Refinement stepCycle: final / Resolution: 2.368→43.235 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8072 0 64 177 8313
Biso mean--87.65 53.72 -
Num. residues----1066
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0058376
X-RAY DIFFRACTIONf_angle_d0.81311423
X-RAY DIFFRACTIONf_chiral_restr0.051267
X-RAY DIFFRACTIONf_plane_restr0.0051471
X-RAY DIFFRACTIONf_dihedral_angle_d7.9855968
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.368-2.40490.37161410.3191247294
2.4049-2.44440.30821380.281269899
2.4444-2.48650.35471540.2771264299
2.4865-2.53170.29261150.2634270699
2.5317-2.58040.27731400.2599266799
2.5804-2.63310.31921440.2488268899
2.6331-2.69030.3221480.2372267999
2.6903-2.75290.2871270.2306269199
2.7529-2.82170.27731230.2323269199
2.8217-2.8980.26531470.2308269699
2.898-2.98320.27081400.23192698100
2.9832-3.07950.31561280.22612723100
3.0795-3.18950.26491440.2115269399
3.1895-3.31720.27211570.20852686100
3.3172-3.46810.25221340.19842716100
3.4681-3.65090.2471350.19952674100
3.6509-3.87950.23461460.17652725100
3.8795-4.17880.19321530.1562718100
4.1788-4.59890.1431580.13472688100
4.5989-5.26330.16571270.13772757100
5.2633-6.62740.1991570.16942723100
6.6274-43.2350.17241600.1672276699
Refinement TLS params.Method: refined / Origin x: 2.708 Å / Origin y: 41.2579 Å / Origin z: 76.4311 Å
111213212223313233
T0.3131 Å2-0.0038 Å20.0283 Å2-0.3638 Å2-0.007 Å2--0.3731 Å2
L0.1557 °20.157 °20.2013 °2-0.3468 °20.4178 °2--0.7049 °2
S0.0902 Å °0.0394 Å °-0.0242 Å °-0.0029 Å °0.012 Å °-0.0311 Å °0.0011 Å °0.004 Å °-0.0894 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allH1 - 218
2X-RAY DIFFRACTION1allH301
3X-RAY DIFFRACTION1allL1 - 218
4X-RAY DIFFRACTION1allL219
5X-RAY DIFFRACTION1allL220
6X-RAY DIFFRACTION1allA1 - 223
7X-RAY DIFFRACTION1allB1 - 300
8X-RAY DIFFRACTION1allC333 - 540
9X-RAY DIFFRACTION1allC541
10X-RAY DIFFRACTION1allS1 - 183

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