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- PDB-7x94: The SARS-CoV-2 receptor binding domain bound with the Fab fragmen... -

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Basic information

Entry
Database: PDB / ID: 7x94
TitleThe SARS-CoV-2 receptor binding domain bound with the Fab fragment of a human neutralizing antibody Ab712
Components
  • Ab712 heavy chain
  • Ab712 light chain
  • Spike glycoproteinSpike protein
KeywordsVIRAL PROTEIN / severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike trimer / COVID-19 / human neutralizing antibody / RBD
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 / Attachment and Entry / membrane fusion / 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 / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / 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 speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsKamada, K. / Shirouzu, M.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21ym0126022 Japan
CitationJournal: iScience / Year: 2023
Title: Potent neutralizing broad-spectrum antibody against SARS-CoV-2 generated from dual-antigen-specific B cells from convalescents.
Authors: Masaru Takeshita / Hidehiro Fukuyama / Katsuhiko Kamada / Takehisa Matsumoto / Chieko Makino-Okamura / Qingshun Lin / Machie Sakuma / Eiki Kawahara / Isato Yamazaki / Tomomi Uchikubo-Kamo / ...Authors: Masaru Takeshita / Hidehiro Fukuyama / Katsuhiko Kamada / Takehisa Matsumoto / Chieko Makino-Okamura / Qingshun Lin / Machie Sakuma / Eiki Kawahara / Isato Yamazaki / Tomomi Uchikubo-Kamo / Yuri Tomabechi / Kazuharu Hanada / Tamao Hisano / Saya Moriyama / Yoshimasa Takahashi / Mutsumi Ito / Masaki Imai / Tadashi Maemura / Yuri Furusawa / Seiya Yamayoshi / Yoshihiro Kawaoka / Mikako Shirouzu / Makoto Ishii / Hideyuki Saya / Yasushi Kondo / Yuko Kaneko / Katsuya Suzuki / Koichi Fukunaga / Tsutomu Takeuchi / /
Abstract: Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing ...Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing antibodies from B cells of convalescents, by using two types of receptor-binding domains, Wuhan strain and the Gamma variant as bait. From 172 antibodies generated, six antibodies neutralized all strains prior to the Omicron variant, and the five antibodies were able to neutralize some of the Omicron sub-strains. Structural analysis showed that these antibodies have a variety of characteristic binding modes, such as ACE2 mimicry. We subjected a representative antibody to the hamster infection model after introduction of the N297A modification, and observed a dose-dependent reduction of the lung viral titer, even at a dose of 2 mg/kg. These results demonstrated that our antibodies have certain antiviral activity as therapeutics, and highlighted the importance of initial cell-screening strategy for the efficient development of therapeutic antibodies.
History
DepositionMar 15, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein
H: Ab712 heavy chain
L: Ab712 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,6944
Polymers196,1073
Non-polymers5871
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 141459.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: From aa1209, additional tags are added at the C-terminal, with Foldon sequence, TEV(tobacco etch virus) protease recognition and cleavage site, AviTag(peptide that allows for enzymatic ...Details: From aa1209, additional tags are added at the C-terminal, with Foldon sequence, TEV(tobacco etch virus) protease recognition and cleavage site, AviTag(peptide that allows for enzymatic biotinylation),and 6xHis affinity tag.
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Protein Ab712 heavy chain


Mass: 29165.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody Ab712 light chain


Mass: 25482.248 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The SARS-CoV-2 spike protein bound with the Fab fragment of a human neutralizing antibody Ab712
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.56 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris hydrochlorideTris-HClTris1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.84 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansMovie frames/image: 40

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPU2.9image acquisition
4CTFFIND4CTF correction
7Coot0.9.6model fitting
9PHENIX1.19.2-4158model refinement
10RELION3.1.2initial Euler assignment
11RELION3.1.2final Euler assignment
12RELION3.1.2classification
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104910 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Corelation coefficient
Atomic model buildingPDB-ID: 5CCK

5cck

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 69.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00253498
ELECTRON MICROSCOPYf_angle_d0.54354764
ELECTRON MICROSCOPYf_chiral_restr0.0448519
ELECTRON MICROSCOPYf_plane_restr0.0042617
ELECTRON MICROSCOPYf_dihedral_angle_d4.7638503

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