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- EMDB-10696: Ectodomain of X-31 Haemagglutinin at pH 8 -

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Basic information

Entry
Database: EMDB / ID: EMD-10696
TitleEctodomain of X-31 Haemagglutinin at pH 8
Map data
Sample
  • Complex: X-31 Influenza Haemagglutinin
    • Protein or peptide: X-31 Influenza Haemagglutinin HA1
    • Protein or peptide: X-31 Influenza Haemagglutinin HA2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHaemagglutinin / Hemagglutinin / Fusion protein / VIRAL PROTEIN
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
Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Biological speciesunidentified influenza virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsBenton DJ / Rosenthal PB
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001078 United Kingdom
The Francis Crick InstituteFC001143 United Kingdom
CitationJournal: Nature / Year: 2020
Title: Structural transitions in influenza haemagglutinin at membrane fusion pH.
Authors: Donald J Benton / Steven J Gamblin / Peter B Rosenthal / John J Skehel /
Abstract: Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope ...Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope glycoproteins bind the infecting virion to cell-surface receptors and mediate membrane fusion. In the case of influenza, the receptor-binding glycoprotein is the haemagglutinin (HA), and following receptor-mediated uptake of the bound virus by endocytosis, it is the HA that mediates fusion of the virus envelope with the membrane of the endosome. Each subunit of the trimeric HA consists of two disulfide-linked polypeptides, HA1 and HA2. The larger, virus-membrane-distal, HA1 mediates receptor binding; the smaller, membrane-proximal, HA2 anchors HA in the envelope and contains the fusion peptide, a region that is directly involved in membrane interaction. The low pH of endosomes activates fusion by facilitating irreversible conformational changes in the glycoprotein. The structures of the initial HA at neutral pH and the final HA at fusion pH have been investigated by electron microscopy and X-ray crystallography. Here, to further study the process of fusion, we incubate HA for different times at pH 5.0 and directly image structural changes using single-particle cryo-electron microscopy. We describe three distinct, previously undescribed forms of HA, most notably a 150 Å-long triple-helical coil of HA2, which may bridge between the viral and endosomal membranes. Comparison of these structures reveals concerted conformational rearrangements through which the HA mediates membrane fusion.
History
DepositionFeb 25, 2020-
Header (metadata) releaseJun 10, 2020-
Map releaseJun 10, 2020-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y5g
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10696.png

Downloads & links

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Map

FileDownload / File: emd_10696.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 400 pix.
= 436. Å		1.09 Å/pix.
x 400 pix.
= 436. Å		1.09 Å/pix.
x 400 pix.
= 436. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-15.171998 - 19.864184999999999
Average (Standard dev.)0.0029293513 (±0.24498667)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 436.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z436.000436.000436.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-15.17219.8640.003

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Supplemental data

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Mask #1

Fileemd_10696_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10696_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10696_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Sample components

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Entire : X-31 Influenza Haemagglutinin

EntireName: X-31 Influenza Haemagglutinin
Components
  • Complex: X-31 Influenza Haemagglutinin
    • Protein or peptide: X-31 Influenza Haemagglutinin HA1
    • Protein or peptide: X-31 Influenza Haemagglutinin HA2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: X-31 Influenza Haemagglutinin

SupramoleculeName: X-31 Influenza Haemagglutinin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: unidentified influenza virus
Molecular weightTheoretical: 164 KDa

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Macromolecule #1: X-31 Influenza Haemagglutinin HA1

MacromoleculeName: X-31 Influenza Haemagglutinin HA1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: unidentified influenza virus
Molecular weightTheoretical: 34.967324 KDa
Recombinant expressionOrganism: Gallus gallus (chicken)
SequenceString: NSTATLCLGH HAVPNGTLVK TITDDQIEVT NATELVQSSS TGKICNNPHR ILDGIDCTLI DALLGDPHCD VFQNETWDLF VERSKAFSN CYPYDVPDYA SLRSLVASSG TLEFITEGFT WTGVTQNGGS NACKRGPGSG FFSRLNWLTK SGSTYPVLNV T MPNNDNFD ...String:
NSTATLCLGH HAVPNGTLVK TITDDQIEVT NATELVQSSS TGKICNNPHR ILDGIDCTLI DALLGDPHCD VFQNETWDLF VERSKAFSN CYPYDVPDYA SLRSLVASSG TLEFITEGFT WTGVTQNGGS NACKRGPGSG FFSRLNWLTK SGSTYPVLNV T MPNNDNFD KLYIWGIHHP STNQEQTSLY VQASGRVTVS TRRSQQTIIP NIGSRPWVRG LSSRISIYWT IVKPGDVLVI NS NGNLIAP RGYFKMRTGK SSIMRSDAPI DTCISECITP NGSIPNDKPF QNVNKITYGA CPKYVKQNTL KLATGMRNVP E

UniProtKB: Hemagglutinin

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Macromolecule #2: X-31 Influenza Haemagglutinin HA2

MacromoleculeName: X-31 Influenza Haemagglutinin HA2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: unidentified influenza virus
Molecular weightTheoretical: 19.912959 KDa
Recombinant expressionOrganism: Gallus gallus (chicken)
SequenceString:
GLFGAIAGFI ENGWEGMIDG WYGFRHQNSE GTGQAADLKS TQAAIDQING KLNRVIEKTN EKFHQIEKEF SEVEGRIQDL EKYVEDTKI DLWSYNAELL VALENQHTID LTDSEMNKLF EKTRRQLREN AEEMGNGCFK IYHKCDNACI ESIRNGTYDH D VYRDEALN NRFQ

UniProtKB: Hemagglutinin

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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: 9 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration2.5 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: 25mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average exposure time: 60.0 sec. / Average electron dose: 33.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 200000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2ypg


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-6y5g:
Ectodomain of X-31 Haemagglutinin at pH 8

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