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- EMDB-17232: Purified human apoferritin at 2.8 A resolution from tilt series p... -

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Basic information

Entry
Database: EMDB / ID: EMD-17232
TitlePurified human apoferritin at 2.8 A resolution from tilt series processed with TomoBEAR
Map dataFinal map, filtered according to local resolution in RELION, sharpened with a B factor of -70. Nominal apix is 1.378, but was calibrated to 1.331.
Sample
  • Organelle or cellular component: ApoferritinFerritin
Keywordsapoferritin / heavy chain / purified sample / StA / tomography / cryo-ET / TomoBEAR / tilt series / CAFR / automated processing / benchmark / OXIDOREDUCTASE
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 2.8 Å
AuthorsBalyschew N / Yushkevich A / Mikirtumov V / Sanchez RM / Sprink T / Kudryashev M
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)KU 3222/2-1 Germany
German Research Foundation (DFG)KU3222/3-1 Germany
German Research Foundation (DFG)INST 335/588-1 FUGG Germany
Alexander von Humboldt FoundationSofja Kovalevskaja Award to MK Germany
Citation
Journal: Nat Commun / Year: 2023
Title: Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR.
Authors: Nikita Balyschew / Artsemi Yushkevich / Vasilii Mikirtumov / Ricardo M Sanchez / Thiemo Sprink / Mikhail Kudryashev /
Abstract: Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to ...Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR-a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent ("white box") approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.
#1: Journal: bioRxiv / Year: 2023
Title: Streamlined Structure Determination by Cryo-Electron Tomography and Subtomogram Averaging using TomoBEAR
Authors: Balyschew N / Yushkevich A / Mikirtumov V / Sanchez RM / Sprink T / Kudryashev M
History
DepositionApr 27, 2023-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_17232.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map, filtered according to local resolution in RELION, sharpened with a B factor of -70. Nominal apix is 1.378, but was calibrated to 1.331.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 200 pix.
= 266.2 Å
1.33 Å/pix.
x 200 pix.
= 266.2 Å
1.33 Å/pix.
x 200 pix.
= 266.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.331 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.120827526 - 0.21670598
Average (Standard dev.)0.00000018526218 (±0.010606842)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 266.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17232_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_17232_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_17232_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Apoferritin

EntireName: ApoferritinFerritin
Components
  • Organelle or cellular component: ApoferritinFerritin

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Supramolecule #1: Apoferritin

SupramoleculeName: Apoferritin / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 5.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 64000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -5.0 µm / Nominal defocus min: -2.0 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 1 / Average electron dose: 3.67 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 60 / Number images used: 21886
Reference model: bagel with dimensions similar to apoferritin
Method: Template Matching / Software - Name: Dynamo (ver. 0.2.0)
Software - details: Dynamo template matching implemented in TomoBEAR with GPU utilization
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0beta) / Software - details: 4.0-beta-2-commit-ce2e93
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0beta) / Software - details: 4.0-beta-2-commit-ce2e93 / Number subtomograms used: 21068
FSC plot (resolution estimation)

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