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- PDB-5uk0: CryoEM structure of an influenza virus receptor-binding site anti... -

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Basic information

Entry
Database: PDB / ID: 5uk0
TitleCryoEM structure of an influenza virus receptor-binding site antibody-antigen interface - Class 2
Components
  • (Hemagglutinin ...) x 2
  • scFV
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / viral glycoprotein / hemagglutinin / antibody fragment / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / apical plasma membrane / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane
Similarity search - Function
Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Biological speciesInfluenza A virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsLiu, Y. / Pan, J. / Caradonna, T. / Jenni, S. / Raymond, D.D. / Schmidt, A.G. / Harrison, S.C. / Grigorieff, N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI-089618 United States
CitationJournal: J Mol Biol / Year: 2017
Title: CryoEM Structure of an Influenza Virus Receptor-Binding Site Antibody-Antigen Interface.
Authors: Yuhang Liu / Junhua Pan / Simon Jenni / Donald D Raymond / Tim Caradonna / Khoi T Do / Aaron G Schmidt / Stephen C Harrison / Nikolaus Grigorieff /
Abstract: Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray ...Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.
History
DepositionJan 19, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2017Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_id_ASTM ..._citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Experimental preparation
Category: em_sample_support / pdbx_audit_support
Item: _em_sample_support.grid_type / _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-8562
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Hemagglutinin HA1
B: Hemagglutinin HA2
C: Hemagglutinin HA1
D: Hemagglutinin HA2
E: Hemagglutinin HA1
F: Hemagglutinin HA2
G: scFV
H: scFV
I: scFV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)257,49933
Polymers250,4849
Non-polymers7,01524
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42510 Å2
ΔGint-90 kcal/mol
Surface area97620 Å2

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Components

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Hemagglutinin ... , 2 types, 6 molecules ACEBDF

#1: Protein Hemagglutinin HA1


Mass: 36024.344 Da / Num. of mol.: 3 / Fragment: UNP residues 18-339
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Strain: A/Solomon Islands/3/2006(H1N1) / Gene: HA / Plasmid: pFastBac / Details (production host): PFASTBAC / Cell line (production host): HI-5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A7UPX0
#2: Protein Hemagglutinin HA2


Mass: 19841.041 Da / Num. of mol.: 3 / Fragment: UNP residues 344-516
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: A/Solomon Islands/3/2006(H1N1) / Gene: HA / Plasmid: pFASTBAC / Cell line (production host): HI-5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A7UPX0

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Antibody , 1 types, 3 molecules GHI

#3: Antibody scFV


Mass: 27629.385 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pVRC / Cell line (production host): HEK293T / Production host: Homo sapiens (human)

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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-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
#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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


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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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: Influenza-virus hemagglutinin H1 complexed with K1915 single-chain variable-domain fragment
Type: COMPLEX
Details: The complex consists of three hemagglutinin head domains bound to three single-chain variable-domain fragments.
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.32 MDa / Experimental value: NO
Source (natural)Organism: Influenza A virus (A/Solomon Islands/3/2006(H1N1))
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Cell: Hi-5
Buffer solutionpH: 8
Details: Beta-octylglucoside was added to a final concentration of 0.07% w/v to induce more variable particle orientations.
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 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: 18000 X / Calibrated magnification: 30444 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 9000 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 13 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10281
Details: The exposure rate was 8 electrons/physical pixel/second.
Image scansSampling size: 2.5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 38 / Used frames/image: 1-38

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4CTFFIND3CTF correction
7Manual placementmodel fitting
9IMAGICinitial Euler assignment
10FREALIGN9.11final Euler assignment
11FREALIGN9.11classification
12FREALIGN9.113D reconstruction
13PHENIX1.10_2155model refinementphenix.real_space_refine
Image processingDetails: The particle images were normalized to have constant variance and zero average. Movies were processed using Unblur.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 252130
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142314 / Algorithm: FOURIER SPACE
Details: Refinement and classification were limited to 10 Angstrom resolution.
Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: Domains were initially placed manually using Chimera. The HA trimer was from PDB ID 5UGY. Fab was initially obtained using MODELLER, with PDB ID 4K8R as template. The structure was refined using PHENIX.

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