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- PDB-9ml4: Structure of the SARS-CoV-2 Spike 6P in complex with the rabbit M... -

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Basic information

Entry
Database: PDB / ID: 9ml4
TitleStructure of the SARS-CoV-2 Spike 6P in complex with the rabbit M8b-A10 Fab
Components
  • M8b-A10 heavy chain
  • M8b-A10 light chain
  • Spike glycoprotein
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / immune system / neutralizing antibody / IMMUNE SYSTEM-VIRAL PROTEIN complex / VIRAL PROTEIN-IMMUNE SYSTEM complex
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 / 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 / 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
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFan, C. / Bjorkman, P.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01-AI165075 United States
Bill & Melinda Gates Foundation United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Cross-reactive sarbecovirus antibodies induced by mosaic RBD nanoparticles.
Authors: Chengcheng Fan / Jennifer R Keeffe / Kathryn E Malecek / Alexander A Cohen / Anthony P West / Viren A Baharani / Annie V Rorick / Han Gao / Priyanthi N P Gnanapragasam / Semi Rho / Jaasiel ...Authors: Chengcheng Fan / Jennifer R Keeffe / Kathryn E Malecek / Alexander A Cohen / Anthony P West / Viren A Baharani / Annie V Rorick / Han Gao / Priyanthi N P Gnanapragasam / Semi Rho / Jaasiel Alvarez / Luisa N Segovia / Theodora Hatziioannou / Paul D Bieniasz / Pamela J Bjorkman /
Abstract: Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) ...Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs.
History
DepositionDec 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
H: M8b-A10 heavy chain
L: M8b-A10 light chain
M: M8b-A10 heavy chain
N: M8b-A10 light chain
P: M8b-A10 heavy chain
Q: M8b-A10 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)567,50751
Polymers558,2179
Non-polymers9,29142
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / Spike HexaPro (6P) / S glycoprotein / E2 / Peplomer protein


Mass: 139344.438 Da / Num. of mol.: 3 / Mutation: F817P, A892P, A899P, A942P, K986P, V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Protein M8b-A10 heavy chain


Mass: 23624.455 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Homo sapiens (human)
#3: Antibody M8b-A10 light chain


Mass: 23103.348 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Production host: Homo sapiens (human)
#4: 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.: 42
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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
Has ligand of interestY
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1SARS-CoV-2 Spike 6P in complex with M8b-A10 FabCOMPLEX#1-#30MULTIPLE SOURCES
2Severe acute respiratory syndrome coronavirus 2 spike glycoproteinCOMPLEX#11RECOMBINANTstabilized with hexaproline
3M8b-A10 FabCOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.57 MDaNO
210.42 MDaNO
310.05 MDaYES
410.025 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
13Oryctolagus cuniculus (rabbit)9986
22Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMTrisTris1
30.02 %Sodium azideNaN31
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 6777

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3particle selection
2SerialEMimage acquisition
4cryoSPARC4.3CTF correction
7UCSF Chimera1.18model fitting
9cryoSPARC4.3initial Euler assignment
10cryoSPARC4.3final Euler assignment
11cryoSPARC4.3classification
12cryoSPARC4.33D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 950884
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134713 / Symmetry type: POINT
Atomic model buildingB value: 90 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeSource nameTypeInitial refinement model-ID
17UZD

7uzd

17UZDPDBexperimental model
27SC1

7sc1

17SC1PDBexperimental model1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 182.15 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002629907
ELECTRON MICROSCOPYf_angle_d0.588340716
ELECTRON MICROSCOPYf_chiral_restr0.04484755
ELECTRON MICROSCOPYf_plane_restr0.0045211
ELECTRON MICROSCOPYf_dihedral_angle_d9.04494852

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