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- PDB-8dxt: Fab arm of antibody GAR12 bound to the receptor binding domain of... -

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Basic information

Entry
Database: PDB / ID: 8dxt
TitleFab arm of antibody GAR12 bound to the receptor binding domain of SARS-CoV-2.
Components
  • Heavy chain of Fab arm of antibody GAR12
  • Light chain of Fab arm of antibody GAR12
  • Spike protein S1
KeywordsANTIVIRAL PROTEIN / anti-CoV-2 / antibodies / convalescent patients / receptor binding domain.
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 / suppression by virus 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 / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / membrane fusion / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / 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 / identical protein binding / membrane / plasma 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 / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / 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 ...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. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / 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 2 (HR2) region profile. / 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 speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus 2
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.25 Å
AuthorsLangley, D.B. / Christ, D. / Henry, J.Y.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia) 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
DepositionAug 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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
Revision 1.2Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Light chain of Fab arm of antibody GAR12
H: Heavy chain of Fab arm of antibody GAR12
R: Spike protein S1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,6654
Polymers71,0943
Non-polymers5711
Water1,62190
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5850 Å2
ΔGint-14 kcal/mol
Surface area27150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)93.609, 41.954, 183.029
Angle α, β, γ (deg.)90.000, 97.080, 90.000
Int Tables number5
Space group name H-MI121

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Components

#1: Antibody Light chain of Fab arm of antibody GAR12


Mass: 23351.973 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Light chain of Fab arm of antibody GAR12 / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Heavy chain of Fab arm of antibody GAR12


Mass: 24766.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Heavy chain of Fab arm of antibody GAR12, with a 6His tag at the C-terminus
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): ExpiCHO / Production host: Cricetulus griseus (Chinese hamster)
#3: Protein Spike protein S1


Mass: 22975.688 Da / Num. of mol.: 1 / Fragment: receptor binding domain
Source method: isolated from a genetically manipulated source
Details: SARS CoV-2 receptor binding domain residues 333-528 with a 6His tag at the C-terminus
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
#4: 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.: 1
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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 90 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.51 Å3/Da / Density % sol: 50.96 % / Description: Rods
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: An equal volume (2 uL) of protein solution at ~5 mg/mL (in 25 mM Tris (pH 8.0), 200 mM NaCl) was combined with well solution (100 mM ammonium citrate (pH 5.5), 20% (w/v) PEG3350.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 11, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 2.25→48.085 Å / Num. obs: 34125 / % possible obs: 100 % / Redundancy: 6.5 % / Biso Wilson estimate: 51.04 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.06 / Rpim(I) all: 0.025 / Rrim(I) all: 0.065 / Net I/σ(I): 17.9
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
2.25-2.326.50.9292045131500.710.3941.011.9100
9-48.0860.02235145850.9990.010.02467.399

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.58 Å48.08 Å
Translation3.58 Å48.08 Å

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
Aimless0.7.7data scaling
PHASER2.8.3phasing
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB 5i1b (Fab) and generic SARS-CoV-2 RBD

5i1b


Resolution: 2.25→48.085 Å / SU ML: 0.35 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 29.51 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2536 1688 4.95 %
Rwork0.2112 32435 -
obs0.2134 34123 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 154.09 Å2 / Biso mean: 71.7112 Å2 / Biso min: 29.31 Å2
Refinement stepCycle: final / Resolution: 2.25→48.085 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4697 0 38 90 4825
Biso mean--107.04 53.94 -
Num. residues----622
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0094898
X-RAY DIFFRACTIONf_angle_d0.9516689
X-RAY DIFFRACTIONf_chiral_restr0.057750
X-RAY DIFFRACTIONf_plane_restr0.006860
X-RAY DIFFRACTIONf_dihedral_angle_d5.8443860
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.25-2.31620.37731380.29792735100
2.3162-2.3910.35481400.2772639100
2.391-2.47640.32271500.27392673100
2.4764-2.57560.31181500.26232643100
2.5756-2.69280.34951240.2562700100
2.6928-2.83480.28271260.24432713100
2.8348-3.01230.32581430.24912667100
3.0123-3.24490.27221450.24762698100
3.2449-3.57130.24241340.22162693100
3.5713-4.08790.23691350.19592737100
4.0879-5.14930.19321290.15942742100
5.1493-48.0850.23691740.1951279599
Refinement TLS params.Method: refined / Origin x: -0.1506 Å / Origin y: -0.6405 Å / Origin z: -26.8762 Å
111213212223313233
T0.4475 Å2-0.0087 Å2-0.0212 Å2-0.5273 Å2-0.0584 Å2--0.4099 Å2
L2.2952 °2-1.2984 °2-0.5227 °2-1.3316 °20.137 °2--1.0071 °2
S-0.1389 Å °-0.323 Å °-0.0453 Å °0.1289 Å °0.1122 Å °0.1489 Å °-0.0795 Å °-0.3988 Å °0.0289 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allL1 - 211
2X-RAY DIFFRACTION1allH1 - 223
3X-RAY DIFFRACTION1allR334 - 534
4X-RAY DIFFRACTION1allR535 - 536
5X-RAY DIFFRACTION1allS1 - 104

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