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- PDB-8cwj: Fab arms of antibodies 4C12-B12 and CR3022 bound to pangolin rece... -

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Basic information

Entry
Database: PDB / ID: 8cwj
TitleFab arms of antibodies 4C12-B12 and CR3022 bound to pangolin receptor binding domain (pRBD)
Components
  • (Heavy chain of ...) x 2
  • (Light chain of ...) x 2
  • Spike glycoprotein
KeywordsIMMUNE SYSTEM / antibody / pangolin / receptor binding domain / class 5 epitope
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / 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 / host cell plasma membrane / virion membrane / membrane
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 / 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 ...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 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Pangolin coronavirus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.449 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 1.2Nov 20, 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 CR3022 antibody Fab
L: Light chain of CR3022 antibody Fab
G: Spike glycoprotein
A: Heavy chain of CR3022 antibody Fab
B: Light chain of CR3022 antibody Fab
I: Spike glycoprotein
J: Heavy chain of 4C12-B12 antibody Fab
K: Light chain of 4C12-B12 antibody Fab
U: Heavy chain of 4C12-B12 antibody Fab
V: Light chain of 4C12-B12 antibody Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,84939
Polymers239,18010
Non-polymers3,66929
Water6,539363
1
H: Heavy chain of CR3022 antibody Fab
L: Light chain of CR3022 antibody Fab
G: Spike glycoprotein
J: Heavy chain of 4C12-B12 antibody Fab
K: Light chain of 4C12-B12 antibody Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,11218
Polymers119,5905
Non-polymers1,52213
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Heavy chain of CR3022 antibody Fab
B: Light chain of CR3022 antibody Fab
I: Spike glycoprotein
U: Heavy chain of 4C12-B12 antibody Fab
V: Light chain of 4C12-B12 antibody Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,73721
Polymers119,5905
Non-polymers2,14716
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)157.433, 144.097, 168.842
Angle α, β, γ (deg.)90.000, 117.510, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Protein , 1 types, 2 molecules GI

#3: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 22891.547 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pangolin coronavirus / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: A0A7D6TQ96

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Antibody , 4 types, 8 molecules HALBJUKV

#1: Antibody Heavy chain of CR3022 antibody Fab


Mass: 24428.432 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpCHO / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Light chain of CR3022 antibody Fab


Mass: 24289.885 Da / Num. of mol.: 2
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 Heavy chain of 4C12-B12 antibody Fab


Mass: 24514.238 Da / Num. of mol.: 2
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)
#5: Antibody Light chain of 4C12-B12 antibody Fab


Mass: 23466.057 Da / Num. of mol.: 2
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)

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Sugars , 2 types, 4 molecules

#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#8: 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.: 1 / 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 , 3 types, 388 molecules

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


Mass: 92.094 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C3H8O3
#9: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Cl
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 363 / 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: 3.55 Å3/Da / Density % sol: 65.32 % / Description: tiles
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: Equal volumes of protein complex (1:1:1 molar ratio, at ~ 5 mg/mL, in 25 mM Tris (pH 8.0), 200 mM NaCl) were combined with well solution (200 mM sodium citrate (pH 5.6), 12% (w/v) PEG6000).

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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: Nov 23, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9536 Å / Relative weight: 1
ReflectionResolution: 2.449→48.69 Å / Num. obs: 122620 / % possible obs: 100 % / Redundancy: 7 % / Biso Wilson estimate: 55.09 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.11 / Rpim(I) all: 0.044 / Rrim(I) all: 0.119 / Net I/σ(I): 11.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.45-2.497.21.1514403860800.7730.4571.2391.4100
13.41-48.696.80.04151707650.9980.0170.04442.797.8

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.45 Å48.69 Å
Translation2.45 Å48.69 Å

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Processing

Software
NameVersionClassification
PHENIX1.11.1refinement
XDSdata reduction
Aimless0.7.7data scaling
PHASER2.8.3phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: generic RBD and Fab

Resolution: 2.449→48.69 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 27.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2301 6036 4.92 %
Rwork0.1962 116534 -
obs0.1979 122570 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 168.95 Å2 / Biso mean: 70.3295 Å2 / Biso min: 28.27 Å2
Refinement stepCycle: final / Resolution: 2.449→48.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16063 0 228 363 16654
Biso mean--111.79 56.29 -
Num. residues----2121
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00216863
X-RAY DIFFRACTIONf_angle_d0.6423023
X-RAY DIFFRACTIONf_chiral_restr0.0462582
X-RAY DIFFRACTIONf_plane_restr0.0042953
X-RAY DIFFRACTIONf_dihedral_angle_d14.6119982
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
2.449-2.47680.34981720.33273921
2.4768-2.5060.40591890.31593831
2.506-2.53650.37881970.3183912
2.5365-2.56860.34322080.29333846
2.5686-2.60240.31942020.28453895
2.6024-2.63810.31942100.27043827
2.6381-2.67580.30361960.25913902
2.6758-2.71570.32122030.25733885
2.7157-2.75810.31042180.25893805
2.7581-2.80340.30832070.25313888
2.8034-2.85170.28161920.24763845
2.8517-2.90350.26912090.24643884
2.9035-2.95940.26442280.23943885
2.9594-3.01980.29642150.24693856
3.0198-3.08540.30122000.25573862
3.0854-3.15720.2671720.23223897
3.1572-3.23610.26591670.23433924
3.2361-3.32360.30872050.23043906
3.3236-3.42140.23171810.21363892
3.4214-3.53180.24062070.20583882
3.5318-3.6580.23431960.19463856
3.658-3.80440.22622160.19793884
3.8044-3.97740.22671900.18663908
3.9774-4.1870.1951990.16783880
4.187-4.44920.17711970.14423913
4.4492-4.79250.17371960.14253885
4.7925-5.27420.17442080.14273897
5.2742-6.03620.20481850.16153941
6.0362-7.60030.21872300.17343900
7.6003-48.690.17692410.17213925
Refinement TLS params.Method: refined / Origin x: 41.9371 Å / Origin y: 2.277 Å / Origin z: 39.4967 Å
111213212223313233
T0.3887 Å20.0326 Å20.0076 Å2-0.3814 Å2-0.0165 Å2--0.2807 Å2
L0.4456 °2-0.1669 °20.1127 °2-0.4154 °20.0099 °2--0.5095 °2
S0.0218 Å °0.066 Å °-0.0484 Å °-0.0488 Å °-0.0064 Å °0.0127 Å °-0.079 Å °0.075 Å °-0.013 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allH1 - 220
2X-RAY DIFFRACTION1allH301
3X-RAY DIFFRACTION1allL1 - 220
4X-RAY DIFFRACTION1allG333 - 529
5X-RAY DIFFRACTION1allG530 - 533
6X-RAY DIFFRACTION1allG536
7X-RAY DIFFRACTION1allG537 - 539
8X-RAY DIFFRACTION1allA1 - 222
9X-RAY DIFFRACTION1allB1 - 218
10X-RAY DIFFRACTION1allI333 - 529
11X-RAY DIFFRACTION1allI531
12X-RAY DIFFRACTION1allI532
13X-RAY DIFFRACTION1allI533 - 538
14X-RAY DIFFRACTION1allI539 - 541
15X-RAY DIFFRACTION1allI542
16X-RAY DIFFRACTION1allJ1 - 219
17X-RAY DIFFRACTION1allJ301
18X-RAY DIFFRACTION1allJ302
19X-RAY DIFFRACTION1allK1 - 214
20X-RAY DIFFRACTION1allK300
21X-RAY DIFFRACTION1allU1 - 219
22X-RAY DIFFRACTION1allU301
23X-RAY DIFFRACTION1allV1 - 300
24X-RAY DIFFRACTION1allS2 - 422
25X-RAY DIFFRACTION1allC1
26X-RAY DIFFRACTION1allC2
27X-RAY DIFFRACTION1allC3 - 12

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