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- PDB-7zj7: X-31 Hemagglutinin Precursor HA0 at pH 4.8 -

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Basic information

Entry
Database: PDB / ID: 7zj7
TitleX-31 Hemagglutinin Precursor HA0 at pH 4.8
ComponentsHemagglutinin,Fibritin
KeywordsVIRAL PROTEIN / virus envelope / receptor binding / membrane fusion
Function / homology
Function and homology information


viral budding from plasma membrane / virion component / clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / 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 / membrane
Similarity search - Function
Fibritin C-terminal / Fibritin C-terminal region / Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Hemagglutinin / Fibritin
Similarity search - Component
Biological speciesInfluenza A virus
Tequatrovirus T4
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsGarcia-Moro, E. / Rosenthal, P.B.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust219946/Z/19/Z United Kingdom
The Francis Crick Institute United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH.
Authors: Eva Garcia-Moro / Jie Zhang / Lesley J Calder / Nick R Brown / Steven J Gamblin / John J Skehel / Peter B Rosenthal /
Abstract: The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage ...The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.
History
DepositionApr 8, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _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
A: Hemagglutinin,Fibritin
B: Hemagglutinin,Fibritin
C: Hemagglutinin,Fibritin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,24918
Polymers186,0813
Non-polymers7,16815
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18610 Å2
ΔGint16 kcal/mol
Surface area78080 Å2

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Components

#1: Protein Hemagglutinin,Fibritin / Collar protein / Whisker antigen control protein


Mass: 62027.055 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: T4 fibritin foldon attached to the C-terminal of HA0 as a trimerization domain. C-terminal His8-tag for protein purification.,T4 fibritin foldon attached to the C-terminal of HA0 as a ...Details: T4 fibritin foldon attached to the C-terminal of HA0 as a trimerization domain. C-terminal His8-tag for protein purification.,T4 fibritin foldon attached to the C-terminal of HA0 as a trimerization domain. C-terminal His8-tag for protein purification.,T4 fibritin foldon attached to the C-terminal of HA0 as a trimerization domain. C-terminal His8-tag for protein purification.,T4 fibritin foldon attached to the C-terminal of HA0 as a trimerization domain. C-terminal His8-tag for protein purification.
Source: (gene. exp.) Influenza A virus (A/Aichi/2/1968(H3N2)), (gene. exp.) Tequatrovirus T4
Strain: A/Aichi/2/1968 H3N2 / Gene: HA, wac / Details (production host): C-terminal His8-tag / Cell (production host): Kidney (Embryonic) / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P03437, UniProt: P10104
#2: 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.: 6
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
#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 732.682 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1221m-1a_1-5]/1-1-2-3/a4-b1_a6-d1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#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: 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.: 3 / Source method: obtained synthetically / 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
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: X-31 Hemagglutinin Precursor (HA0) / Type: COMPLEX
Details: Uncleaved precursor form of the X-31 influenza hemagglutinin with the T4 fibritin foldon attached to the C-terminus.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Influenza A virus (A/Aichi/2/1968(H3N2))387139
31Enterobacteria phage T4 (virus)10665
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
21Homo sapiens (human)9606Expi293F
31Homo sapiens (human)9606Expi293F
Buffer solutionpH: 4.8
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample protein tended to aggregate and bind to the carbon film. 0.1% b-octyl glucoside was added.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 4s blot

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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: 3300 nm / Nominal defocus min: 1500 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 41.15 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 33977
Details: 15696 images on session 1, 18281 images on session 2.
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.2/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
5RELION3.1.1CTF correction
8Coot0.9.6model fitting
10RELION3.1.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
12cryoSPARC3.1classification
13cryoSPARC3.13D reconstruction
14REFMAC5model refinement
Image processingDetails: Images were subject to whole-frame motion correction and dose weighting.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1235000 / Details: 646k from session 1, 589k from session 2.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149000 / Algorithm: FOURIER SPACE / Details: CryoSPARC Non-uniform Refinement. / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 180 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 6Y5K

6y5k


Accession code: 6Y5K / Source name: PDB / Type: experimental model

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