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- EMDB-51755: Cryo-EM map of revitrified Hemagglutinin -

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Basic information

Entry
Database: EMDB / ID: EMD-51755
TitleCryo-EM map of revitrified Hemagglutinin
Map dataMap of revitrified HA
Sample
  • Complex: Hemagglutinin
KeywordsHemagglutinin / VIRAL PROTEIN
Biological speciesInfluenza A virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsStraub MS / Harder OF / Mowry NJ / Barrass SV / Hruby J / Drabbels M / Lorenz UJ
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation213773 Switzerland
CitationJournal: bioRxiv / Year: 2024
Title: Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation.
Authors: Monique S Straub / Oliver F Harder / Nathan J Mowry / Sarah V Barrass / Jakub Hruby / Marcel Drabbels / Ulrich J Lorenz /
Abstract: Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the ...Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether. We have previously observed that laser flash melting and revitrification of cryo samples reduces preferred orientation for large, symmetric particles. Here, we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.
History
DepositionOct 8, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51755.png

Downloads & links

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Map

FileDownload / File: emd_51755.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of revitrified HA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 360 pix.
= 263.52 Å		0.73 Å/pix.
x 360 pix.
= 263.52 Å		0.73 Å/pix.
x 360 pix.
= 263.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.732 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.157
Minimum - Maximum-0.5345607 - 0.9293786
Average (Standard dev.)-0.00012189349 (±0.02243866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 263.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51755_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B to revitrified HA

Fileemd_51755_half_map_1.map
AnnotationHalf map B to revitrified HA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A to revitrified HA

Fileemd_51755_half_map_2.map
AnnotationHalf map A to revitrified HA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hemagglutinin

EntireName: Hemagglutinin
Components
  • Complex: Hemagglutinin

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Supramolecule #1: Hemagglutinin

SupramoleculeName: Hemagglutinin / type: complex / ID: 1 / Parent: 0 / Details: HA(A/Hong Kong/1/1968)(H3N2)(aa 17-529) Trimer
Source (natural)Organism: Influenza A virus / Strain: H3N2
Molecular weightTheoretical: 170 KDa

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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: 7.5 / Component - Concentration: 1.0 x / Component - Formula: PBS / Component - Name: phosphate buffered saline
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 6476 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
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: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 50000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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