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- EMDB-14743: X-31 Hemagglutinin Precursor HA0 at pH 4.8 -

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Basic information

Entry
Database: EMDB / ID: EMD-14743
TitleX-31 Hemagglutinin Precursor HA0 at pH 4.8
Map dataSharpened map of the X-31 influenza hemagglutinin precursor HA0 at low pH.
Sample
  • Complex: X-31 Hemagglutinin Precursor (HA0)
    • Protein or peptide: Hemagglutinin,Fibritin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 (A/Aichi/2/1968(H3N2)) / Tequatrovirus T4
Methodsingle particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsGarcia-Moro E / Rosenthal PB
Funding support United Kingdom, 2 items
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, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14743.png

Downloads & links

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Map

FileDownload / File: emd_14743.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of the X-31 influenza hemagglutinin precursor HA0 at low pH.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.9696087 - 1.6164511
Average (Standard dev.)0.001566113 (±0.028221076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14743_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map processed with the deepEMhancer software, used to...

Fileemd_14743_additional_1.map
AnnotationMap processed with the deepEMhancer software, used to aid model building.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2.

Fileemd_14743_half_map_1.map
AnnotationHalf map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1.

Fileemd_14743_half_map_2.map
AnnotationHalf map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : X-31 Hemagglutinin Precursor (HA0)

EntireName: X-31 Hemagglutinin Precursor (HA0)
Components
  • Complex: X-31 Hemagglutinin Precursor (HA0)
    • Protein or peptide: Hemagglutinin,Fibritin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: X-31 Hemagglutinin Precursor (HA0)

SupramoleculeName: X-31 Hemagglutinin Precursor (HA0) / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Uncleaved precursor form of the X-31 influenza hemagglutinin with the T4 fibritin foldon attached to the C-terminus.
Source (natural)Organism: Influenza A virus (A/Aichi/2/1968(H3N2))
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Expi293F

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Macromolecule #1: Hemagglutinin,Fibritin

MacromoleculeName: Hemagglutinin,Fibritin / type: protein_or_peptide / ID: 1
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.
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Tequatrovirus T4
Molecular weightTheoretical: 62.027055 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QDLPGNDNST ATLCLGHHAV PNGTLVKTIT DDQIEVTNAT ELVQSSSTGK ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVE RSKAFSNCYP YDVPDYASLR SLVASSGTLE FITEGFTWTG VTQNGGSNAC KRGPGSGFFS RLNWLTKSGS T YPVLNVTM ...String:
QDLPGNDNST ATLCLGHHAV PNGTLVKTIT DDQIEVTNAT ELVQSSSTGK ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVE RSKAFSNCYP YDVPDYASLR SLVASSGTLE FITEGFTWTG VTQNGGSNAC KRGPGSGFFS RLNWLTKSGS T YPVLNVTM PNNDNFDKLY IWGIHHPSTN QEQTSLYVQA SGRVTVSTRR SQQTIIPNIG SRPWVRGLSS RISIYWTIVK PG DVLVINS NGNLIAPRGY FKMRTGKSSI MRSDAPIDTC ISECITPNGS IPNDKPFQNV NKITYGACPK YVKQNTLKLA TGM RNVPEK QTRGLFGAIA GFIENGWEGM IDGWYGFRHQ NSEGTGQAAD LKSTQAAIDQ INGKLNRVIE KTNEKFHQIE KEFS EVEGR IQDLEKYVED TKIDLWSYNA ELLVALENQH TIDLTDSEMN KLFEKTRRQL RENAEEMGNG CFKIYHKCDN ACIES IRNG TYDHDVYRDE ALNNRFQIKG GGRENLYFQG GGGSGYIPEA PRDGQAYVRK DGEWVLLSTF LGHHHHHHHH

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 4.8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4s blot.
DetailsThe sample protein tended to aggregate and bind to the carbon film. 0.1% b-octyl glucoside was added.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-32 / Number grids imaged: 2 / Number real images: 33977 / Average exposure time: 8.0 sec. / Average electron dose: 41.15 e/Å2
Details: 15696 images on session 1, 18281 images on session 2.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1235000 / Details: 646k from session 1, 589k from session 2.
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.13), RELION (ver. 3.1.1))
Startup modelType of model: NONE / Details: RELION 3D initial model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.1)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Details: CryoSPARC Non-uniform Refinement. / Number images used: 149000
DetailsImages were subject to whole-frame motion correction and dose weighting.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6y5k

RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 180
Output model

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

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