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- PDB-8f38: Cryo-EM structure of X6 COBRA (H1N1) hemagglutinin bound to CR6261 Fab -

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Basic information

Entry
Database: PDB / ID: 8f38
TitleCryo-EM structure of X6 COBRA (H1N1) hemagglutinin bound to CR6261 Fab
Components
  • CR6261 Fab heavy chain
  • CR6261 Fab light chain
  • hemagglutinin
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / NIAID hemagglutinin stalk binding antibody / Structural Genomics / Seattle Structural Genomics Center for Infectious Disease / SSGCID / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsSeattle Structural Genomics Center for Infectious Disease (SSGCID)
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)HHSN272201700059C United States
CitationJournal: Structure / Year: 2024
Title: Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6.
Authors: Kaito A Nagashima / John V Dzimianski / Meng Yang / Jan Abendroth / Giuseppe A Sautto / Ted M Ross / Rebecca M DuBois / Thomas E Edwards / Jarrod J Mousa /
Abstract: Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been ...Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.
History
DepositionNov 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Aug 28, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: CR6261 Fab light chain
D: CR6261 Fab heavy chain
E: CR6261 Fab heavy chain
F: CR6261 Fab heavy chain
A: hemagglutinin
B: hemagglutinin
C: hemagglutinin
H: CR6261 Fab light chain
I: CR6261 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)340,73133
Polymers333,7179
Non-polymers7,01524
Water90150
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 6 molecules GHIDEF

#1: Antibody CR6261 Fab light chain


Mass: 23257.783 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#2: Antibody CR6261 Fab heavy chain


Mass: 24810.018 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Protein / Non-polymers , 2 types, 53 molecules ABC

#3: Protein hemagglutinin


Mass: 63171.098 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: X6 COBRA (H1N1) hemagglutinin construct / Source: (gene. exp.) synthetic construct (others) / Production host: Trichoplusia ni (cabbage looper)
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 50 / Source method: isolated from a natural source / Formula: H2O

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Sugars , 3 types, 24 molecules

#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.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: 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.: 18
Source method: isolated from a genetically manipulated source
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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Details

Has ligand of interestN
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

ComponentName: Cryo-EM structure of X6 COBRA (H1N1) hemagglutinin bound to CR6261 Fab
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.45 MDa / Experimental value: NO
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
225 mMTris(hydroxymethyl)aminomethane hydrochloride1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / 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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8973

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Processing

SoftwareName: PHENIX / Version: dev_4274: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2.2.0particle selection
2SerialEMimage acquisition
4cryoSPARC2.2.0CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10cryoSPARC2.2.0initial Euler assignment
11cryoSPARC2.2.0final Euler assignment
13cryoSPARC2.2.03D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 385474
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 385474 / Symmetry type: POINT
Atomic model buildingB value: 115.1 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6UYN

6uyn


Accession code: 6UYN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317307
ELECTRON MICROSCOPYf_angle_d0.51323514
ELECTRON MICROSCOPYf_dihedral_angle_d6.752502
ELECTRON MICROSCOPYf_chiral_restr0.0442640
ELECTRON MICROSCOPYf_plane_restr0.0053003

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