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- EMDB-33833: In situ subtomogram average of f-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-33833
TitleIn situ subtomogram average of f-actin
Map data
Sample
  • Complex: f-actin bound by tropomyosin
Keywordsfilamentous / f-actin / CYTOSOLIC PROTEIN
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 12.1 Å
AuthorsEisenstein F / Danev R
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)P20764 Japan
Japan Science and Technology18069571 Japan
CitationJournal: Nat Methods / Year: 2023
Title: Parallel cryo electron tomography on in situ lamellae.
Authors: Fabian Eisenstein / Haruaki Yanagisawa / Hiroka Kashihara / Masahide Kikkawa / Sachiko Tsukita / Radostin Danev /
Abstract: In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native ...In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.
History
DepositionJul 14, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33833.png

Downloads & links

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Map

FileDownload / File: emd_33833.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
3.28 Å/pix.
x 100 pix.
= 328. Å		3.28 Å/pix.
x 100 pix.
= 328. Å		3.28 Å/pix.
x 100 pix.
= 328. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-3.087966 - 12.371726000000001
Average (Standard dev.)0.10754641 (±1.1672889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_33833_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Half map: #2

Fileemd_33833_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : f-actin bound by tropomyosin

EntireName: f-actin bound by tropomyosin
Components
  • Complex: f-actin bound by tropomyosin

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Supramolecule #1: f-actin bound by tropomyosin

SupramoleculeName: f-actin bound by tropomyosin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: EpH4

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Homemade / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 80 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 0.5 sec. / Average electron dose: 3.63 e/Å2
Details: Tilt series were recorded in parallel using beam image shift
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: 5.0 µm / Nominal defocus min: 5.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.88 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.87 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 12.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number subtomograms used: 4254
ExtractionNumber tomograms: 4 / Number images used: 13541
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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