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- EMDB-8731: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstro... -

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Basic information

Entry
Database: EMDB / ID: EMD-8731
TitleInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees
Map dataInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, sharpened using a B-factor of 320
Sample
  • Organelle or cellular component: Influenza hemagglutinin (HA) trimer
    • Protein or peptide: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2
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 speciesInfluenza A virus (A/Hong Kong/1/1968(H3N2))
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsTan Y / Baldwin PR / Potter CS / Carragher B / Lyumkis D
Funding support Singapore, United States, 8 items
OrganizationGrant numberCountry
Agency for Science, Technology and Research Singapore Singapore
US National Institutes of Health (NIH)DP5 OD021396-01 United States
Leona M. and Harry B. Helmsley Charitable Trust Grant2017-PG-MED001 United States
National Institute of Aging K99 transitional awardAG050749 United States
Jane Coffin Childs Foundation post-doctoral fellowship United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM053757 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Simons Foundation349247 United States
CitationJournal: Nat Methods / Year: 2017
Title: Addressing preferred specimen orientation in single-particle cryo-EM through tilting.
Authors: Yong Zi Tan / Philip R Baldwin / Joseph H Davis / James R Williamson / Clinton S Potter / Bridget Carragher / Dmitry Lyumkis /
Abstract: We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the ...We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.
History
DepositionMay 14, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseJun 14, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0367
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0367
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8731.png

Downloads & links

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Map

FileDownload / File: emd_8731.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, sharpened using a B-factor of 320
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0367 / Movie #1: 0.0367
Minimum - Maximum-0.071533866 - 0.13927907
Average (Standard dev.)-0.00001959061 (±0.004145391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0720.139-0.000

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Supplemental data

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Mask #1

Fileemd_8731_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_additional_1.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_additional_2.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, map-to-model 3DFSC, thresholded at 0.5
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_additional_3.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, half-map 3DFSC, thresholded at 0.143
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_additional_4.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, map-to-model 3DFSC, raw
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_additional_5.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, half-map 3DFSC, raw
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_half_map_1.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Influenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom...

Fileemd_8731_half_map_2.map
AnnotationInfluenza hemagglutinin (HA) trimer reconstruction at 4.2 Angstrom resolution using particles from micrographs tilted at 40 degrees, half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Influenza hemagglutinin (HA) trimer

EntireName: Influenza hemagglutinin (HA) trimer
Components
  • Organelle or cellular component: Influenza hemagglutinin (HA) trimer
    • Protein or peptide: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2

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Supramolecule #1: Influenza hemagglutinin (HA) trimer

SupramoleculeName: Influenza hemagglutinin (HA) trimer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: A/Hong Kong/1/1968 (H3N2), obtained commercially from MyBioSource, catalog MBS434205
Source (natural)Organism: Influenza A virus (A/Hong Kong/1/1968(H3N2))
Molecular weightTheoretical: 150 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293

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Macromolecule #1: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2

MacromoleculeName: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Hong Kong/1/1968(H3N2))
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QDLPGNDNST ATLCLGHHAV PNGTLVKTIT DDQIEVTNAT ELVQSSSTGK ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVER SKAFSNCYPY DVPDYASLRS LVASSGTLEF ITEGFTWTGV TQNGGSNACK RGPGSGFFSR LNWLTKSGST YPVLNVTMPN ...String:
QDLPGNDNST ATLCLGHHAV PNGTLVKTIT DDQIEVTNAT ELVQSSSTGK ICNNPHRILD GIDCTLIDAL LGDPHCDVFQ NETWDLFVER SKAFSNCYPY DVPDYASLRS LVASSGTLEF ITEGFTWTGV TQNGGSNACK RGPGSGFFSR LNWLTKSGST YPVLNVTMPN NDNFDKLYIW GVHHPSTNQE QTSLYVQASG RVTVSTRRSQ QTIIPNIGSR PWVRGLSSRI SIYWTIVKPG DVLVINSNGN LIAPRGYFKM RTGKSSIMRS DAPIDTCISE CITPNGSIPN DKPFQNVNKI TYGACPKYVK QNTLKLATGM RNVPEKQTRG LFGAIAGFIE NGWEGMIDGW YGFRHQNSEG TGQAADLKST QAAIDQINGK LNRVIEKTNE KFHQIEKEFS EVEGRIQDLE KYVEDTKIDL WSYNAELLVA LENQHTIDLT DSEMNKLFEK TRRQLRENAE DMGNGCFKIY HKCDNACIES IRNGTYDHDV YRDEALNNRF QIKGVELKSG YKD

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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

Concentration0.75 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
137.0 mMNaClSodium Chloride
2.7 mMKClPotassium Chloride
10.0 mMNa2HPO4Disodium Phosphate
1.8 mMKH2PO4Monopotassium Phosphate

Details: PBS Buffer
GridModel: C-Flat CF-1.2/1.3-4Au / Material: GOLD / Mesh: 400 / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3
Details: 3 microliters of 0.75 mg/mL sample was added to a plasma-cleaned (Gatan Solarus) 1.2-micron-hole, 1.3-micron-spacing holey gold grid (made in-house) and plunge-frozen in liquid ethane using ...Details: 3 microliters of 0.75 mg/mL sample was added to a plasma-cleaned (Gatan Solarus) 1.2-micron-hole, 1.3-micron-spacing holey gold grid (made in-house) and plunge-frozen in liquid ethane using the Cryoplunge 3 system (Gatan) operating at 80% humidity, 298K ambient temperature..
DetailsFraction was obtained directly from a sucrose gradient, and was spin-concentrated in a 100 kDa MW-cutoff filter.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsIn order to account for highly preferred orientations of the specimen, data were acquired using tilts of 40 degrees. The CTF was estimated on a per-particle basis to account for the gradient of CTF values across individual micrographs.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-100 / Number grids imaged: 2 / Number real images: 847 / Average exposure time: 20.0 sec. / Average electron dose: 82.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
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

Particle selectionNumber selected: 287820
Details: 287820 particles were picked using Gautomatch with templates made from an initial 2D classification of a subset of the particles. These particles were put through 2D classification to produce 158432 particles.
CTF correctionSoftware - Name: Gctf (ver. 0.5)
Startup modelType of model: INSILICO MODEL
In silico model: Ab initio model was produced using CryoSPARC using the 158432 particles.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2)
Details: Directional anisotropy is present in the resolution, with a range from 3.7 to 5.5 Angstrom, 3D FSC sphericity of 0.97, and map-to-model resolution of 4.4 Angstrom.
Number images used: 158432
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)

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