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- PDB-8ghk: CryoEM structure of Influenza A virus A/Melbourner/1/1946 (H1N1) ... -

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Basic information

Entry
Database: PDB / ID: 8ghk
TitleCryoEM structure of Influenza A virus A/Melbourner/1/1946 (H1N1) hemagglutinin bound to GS10-X6-BE4 Fab
Components
  • (GS10-X6-BE4 Fab ...) x 2
  • 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 speciesInfluenza A virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 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)75N93022C00036 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
DepositionMar 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hemagglutinin
B: Hemagglutinin
C: Hemagglutinin
H: GS10-X6-BE4 Fab Heavy chain
L: GS10-X6-BE4 Fab light chain
M: GS10-X6-BE4 Fab Heavy chain
N: GS10-X6-BE4 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,77919
Polymers222,1917
Non-polymers3,58812
Water00
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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Protein , 1 types, 3 molecules ABC

#1: Protein Hemagglutinin


Mass: 53994.246 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: 1/Melbourne/1/1946(H1N1) / Gene: HA / Plasmid: InvcI.18715.a.K1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)

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Antibody , 2 types, 4 molecules HMLN

#2: Antibody GS10-X6-BE4 Fab Heavy chain


Mass: 16154.263 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / Strain (production host): HEK293e
#3: Antibody GS10-X6-BE4 Fab light chain


Mass: 13949.619 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / Strain (production host): HEK293e

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Sugars , 3 types, 12 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.: 2
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.: 1
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.: 9
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
Sequence detailsThe full sequences of the Fab chains were not provided by the depositors; only the sequences of the ...The full sequences of the Fab chains were not provided by the depositors; only the sequences of the variable domain portions are presented.

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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: CryoEM structure of Influenza A virus A/Melbourner/1/1946 (H1N1) hemagglutinin bound to GS10-X6-BE4 Fab
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.221 MDa / Experimental value: NO
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
1150 mMsodium chloride1
225 mMTris/HCl1
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: C-flat-2/1
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 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7031

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Processing

SoftwareName: PHENIX / Version: dev_4788: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.1particle selection
2SerialEMimage acquisition
4cryoSPARC3.1CTF correction
7UCSF ChimeraXmodel fitting
8Coot0.9.8.7model fitting
10PHENIX1.20.1-4788model refinement
11cryoSPARC3.1initial Euler assignment
14cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100708 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8DIS

8dis


Accession code: 8DIS / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411953
ELECTRON MICROSCOPYf_angle_d0.54216335
ELECTRON MICROSCOPYf_dihedral_angle_d4.6921788
ELECTRON MICROSCOPYf_chiral_restr0.0481900
ELECTRON MICROSCOPYf_plane_restr0.0042078

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