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- PDB-6wxb: Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrifie... -

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Basic information

Entry
Database: PDB / ID: 6wxb
TitleCryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrified Using Back-it-up
ComponentsHemagglutinin
KeywordsVIRAL PROTEIN / Hemagglutinin / trimer / Influenza / back-it-up / through-grid wicking
Function / homology
Function and homology information


viral budding from plasma membrane / 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
Hemagglutinin (Ha1 Chain); Chain: A; domain 1 / Haemagglutinin, alpha/beta domain, HA1 chain / Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
beta-D-mannopyranose / Hemagglutinin
Similarity search - Component
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTan, Y.Z. / Rubinstein, J.L.
Funding support Canada, 3items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canada Excellence Research Chair Award Canada
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: Through-grid wicking enables high-speed cryoEM specimen preparation.
Authors: Yong Zi Tan / John L Rubinstein /
Abstract: Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, ...Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
History
DepositionMay 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.2Nov 18, 2020Group: Database references / Structure summary / Category: chem_comp / citation
Item: _chem_comp.pdbx_synonyms / _citation.journal_abbrev ..._chem_comp.pdbx_synonyms / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Hemagglutinin
B: Hemagglutinin
C: Hemagglutinin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,76118
Polymers188,5663
Non-polymers3,19515
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Hemagglutinin


Mass: 62855.215 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Strain: A/Hong Kong/1/1968 H3N2 / Gene: HA / Production host: Homo sapiens (human) / Strain (production host): HEK293S / References: UniProt: Q91MA7
#2: 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.: 12
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
#3: Sugar ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DManpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-mannopyranoseCOMMON NAMEGMML 1.0
b-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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 Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)Organism: H3N2 subtype (virus)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was glow discharged on both sides for 120s each.
Grid material: COPPER/RHODIUM / Grid type: Homemade
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE / Humidity: 50 % / Chamber temperature: 298 K
Details: Back-it-up (ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device) was used

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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 / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1556
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2EPU2.6image acquisition
4cryoSPARC2CTF correction
7UCSF Chimera1.13model fitting
9cryoSPARC2initial Euler assignment
10cryoSPARC2final Euler assignment
12cryoSPARC23D reconstruction
13Coot0.8model refinement
14PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 378143
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128305 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3WHE

3whe

RefinementCross valid method: NONE / Stereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007811916
ELECTRON MICROSCOPYf_angle_d1.007116197
ELECTRON MICROSCOPYf_chiral_restr0.08021818
ELECTRON MICROSCOPYf_plane_restr0.00622106
ELECTRON MICROSCOPYf_dihedral_angle_d11.08267083

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