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- EMDB-0025: Influenza hemagglutinin (HA) trimer reconstruction at 3.2 Angstro... -

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Basic information

Entry
Database: EMDB / ID: EMD-0025
TitleInfluenza hemagglutinin (HA) trimer reconstruction at 3.2 Angstrom resolution using data from EMPIAR-10097 processed with Warp and cryoSPARC
Map data
Sample
  • Complex: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2
    • Protein or peptide: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2
Biological speciesInfluenza A virus (A/Hong Kong/1/1968(H3N2))
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsTegunov D / Cramer P
Funding support Germany, 3 items
OrganizationGrant numberCountry
European Research Council693023 Germany
Volkswagen Foundation Germany
German Research FoundationSFB 860 Germany
Citation
Journal: Nat Methods / Year: 2019
Title: Real-time cryo-electron microscopy data preprocessing with Warp.
Authors: Dimitry Tegunov / Patrick Cramer /
Abstract: The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we ...The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we describe Warp, a software that automates all preprocessing steps of cryo-EM data acquisition and enables real-time evaluation. Warp corrects micrographs for global and local motion, estimates the local defocus and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. The software further includes deep-learning-based models for accurate particle picking and image denoising. The output from Warp can be fed into established programs for particle classification and 3D-map refinement. Our benchmarks show improvement in the nominal resolution, which went from 3.9 Å to 3.2 Å, of a published cryo-EM data set for influenza virus hemagglutinin. Warp is easy to install from http://github.com/cramerlab/warp and computationally inexpensive, and has an intuitive, streamlined user interface.
#1: Journal: Biorxiv / Year: 2018
Title: Real-time cryo-EM data pre-processing with Warp
Authors: Tegunov D / Cramer P
History
DepositionMay 22, 2018-
Header (metadata) releaseJun 27, 2018-
Map releaseJun 27, 2018-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0025.png

Downloads & links

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Map

FileDownload / File: emd_0025.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-4.044406 - 6.6609964
Average (Standard dev.)-0.00011054852 (±0.16333474)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-4.0446.661-0.000

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Supplemental data

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Mask #1

Fileemd_0025_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_0025_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_0025_half_map_2.map
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 A/Hong Kong/1/1968 H3N2

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

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

SupramoleculeName: Influenza hemagglutinin (HA) Trimer A/Hong Kong/1/1968 H3N2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Influenza A virus (A/Hong Kong/1/1968(H3N2))
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293
Molecular weightTheoretical: 150 KDa

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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
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 / Details: Same as EMD-8731
GridModel: C-flat-1.2/1.3 4C / Material: GOLD / Mesh: 400 / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Details: Same as EMD-8731
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3 / Details: Same as EMD-8731.
DetailsSame as EMD-8731

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 FIELDBright-field microscopy / 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
DetailsSame as EMD-8731
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 / Number grids imaged: 2 / Number real images: 847 / Average exposure time: 20.0 sec. / Average electron dose: 82.0 e/Å2 / Details: Same as EMD-8731
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 489215
CTF correctionSoftware - Name: Warp / Software - details: 6x6x1 model resolution
Details: On-the-fly correction in cryoSPARC, based on local defocus values from Warp
Startup modelType of model: NONE / Details: 6 ab initio models created in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 6 / Avg.num./class: 81500 / Software - Name: cryoSPARC
Details: 3D classification initialized with the 6 ab initio models obtained earlier.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Mask used for FSC automatically generated in cryoSPARC.
Number images used: 249495
FSC plot (resolution estimation)

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