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- EMDB-21954: Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrifie... -

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Basic information

Entry
Database: EMDB / ID: EMD-21954
TitleCryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrified Using Back-it-up
Map dataSharpened map
Sample
  • Organelle or cellular component: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)
    • Protein or peptide: Hemagglutinin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: beta-D-mannopyranose
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 speciesH3N2 subtype (virus) / Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTan YZ / Rubinstein JL
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Canada Excellence Research Chair Award Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) 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, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wxb
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21954.png

Downloads & links

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Map

FileDownload / File: emd_21954.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-7.2507486 - 11.195214
Average (Standard dev.)-0.000964305 (±0.29797086)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z263.680263.680263.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-7.25111.195-0.001

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Supplemental data

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Mask #1

Fileemd_21954_msk_1.map
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AxesZYX

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Additional map: 3DFSC - Raw

Fileemd_21954_additional_1.map
Annotation3DFSC - Raw
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Additional map: 3DFSC - Thresholded

Fileemd_21954_additional_2.map
Annotation3DFSC - Thresholded
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Additional map: 3DFSC - Thresholded and Binarized

Fileemd_21954_additional_3.map
Annotation3DFSC - Thresholded and Binarized
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Additional map: Local resolution map

Fileemd_21954_additional_4.map
AnnotationLocal resolution map
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Additional map: Raw map

Fileemd_21954_additional_5.map
AnnotationRaw map
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Half map: Half map 1

Fileemd_21954_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_21954_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)

EntireName: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)
Components
  • Organelle or cellular component: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)
    • Protein or peptide: Hemagglutinin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: beta-D-mannopyranose

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Supramolecule #1: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)

SupramoleculeName: Influenza Hemagglutinin Trimer (H3N2) (A/Hong Kong/1/1968)
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: H3N2 subtype (virus)
Molecular weightTheoretical: 190 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F

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Macromolecule #1: Hemagglutinin

MacromoleculeName: Hemagglutinin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Strain: A/Hong Kong/1/1968 H3N2
Molecular weightTheoretical: 62.855215 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 IWGVHHPSTN 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 RENAEDMGNG CFKIYHKCDN ACIES IRNG TYDHDVYRDE ALNNRFQIKG VELKSGYDGG GGTGGGGTGR MKQIEDKIEE ILSKIYHIEN EIARIKKLIG ERHHHH HH

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 12 / 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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Macromolecule #3: beta-D-mannopyranose

MacromoleculeName: beta-D-mannopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: BMA
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BMA:
beta-D-mannopyranose / Mannose

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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

BufferpH: 7.4
GridModel: Homemade / Material: COPPER/RHODIUM / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Grid was glow discharged on both sides for 120s each.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 50 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 1556 / Average exposure time: 9.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 378143
CTF correctionSoftware - Name: cryoSPARC (ver. 2)
Startup modelType of model: INSILICO MODEL
In silico model: Made using cryoSPARC 2 ab initio refinement
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 128305
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3whe

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6wxb:
Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrified Using Back-it-up

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