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- EMDB-52722: Human gamma-tubulin ring complex in centrosomes of HCT116 and HeL... -

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Entry
Database: EMDB / ID: EMD-52722
TitleHuman gamma-tubulin ring complex in centrosomes of HCT116 and HeLa cells
Map dataSubtomogram average of human gamma-TuRC in centrosomes of HeLa and HCT116 cells
Sample
  • Cell: gamma-tubulin ring complex in human centrosomes of HCT116 and HeLa cells
Keywordscentrosome / cytoskeleton / microtubule / microtubule nucleation / complex / template / cap / gamma-tubulin / gamma-tubulin ring complex / cell cycle / nedd1 / neural precursor cell-expressed developmentally down-regulated 1 / CDK5RAP2 / cyclin-dependent kinase 5 regulatory subunit associated protein 2
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 30.6 Å
AuthorsHofer FW / Pfeffer S
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)DFG Schi 295/4-4 Germany
German Research Foundation (DFG)DFG SCHI 295/11-1 Germany
German Research Foundation (DFG)DFG PF 963/1-4 Germany
German Research Foundation (DFG)DFG PF 963/4-1 Germany
CitationJournal: Nat Methods / Year: 2023
Title: ELI trifocal microscope: a precise system to prepare target cryo-lamellae for in situ cryo-ET study.
Authors: Shuoguo Li / Ziyan Wang / Xing Jia / Tongxin Niu / Jianguo Zhang / Guoliang Yin / Xiaoyun Zhang / Yun Zhu / Gang Ji / Fei Sun /
Abstract: Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo- ...Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent cryo-ET. Here we describe a precise cryogenic fabrication system, ELI-TriScope, which sets electron (E), light (L) and ion (I) beams at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella. ELI-TriScope uses a commercial dual-beam scanning electron microscope modified to incorporate a cryo-holder-based transfer system and embed an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome with a success rate of ~91% and discovered new in situ structural features of the human centrosome by cryo-ET.
History
DepositionFeb 4, 2025-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52722.png

Downloads & links

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Map

FileDownload / File: emd_52722.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of human gamma-TuRC in centrosomes of HeLa and HCT116 cells
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.3 Å/pix.
x 128 pix.
= 550.592 Å		4.3 Å/pix.
x 128 pix.
= 550.592 Å		4.3 Å/pix.
x 128 pix.
= 550.592 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.3015 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.56
Minimum - Maximum-0.2655811 - 0.8887879
Average (Standard dev.)0.018088784 (±0.11172859)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 550.592 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map for subtomogram average of human gamma-TuRC...

Fileemd_52722_half_map_1.map
AnnotationHalf map for subtomogram average of human gamma-TuRC in centrosomes of HeLa and HCT116 cells
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map for subtomogram average of human gamma-TuRC...

Fileemd_52722_half_map_2.map
AnnotationHalf map for subtomogram average of human gamma-TuRC in centrosomes of HeLa and HCT116 cells
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : gamma-tubulin ring complex in human centrosomes of HCT116 and HeL...

EntireName: gamma-tubulin ring complex in human centrosomes of HCT116 and HeLa cells
Components
  • Cell: gamma-tubulin ring complex in human centrosomes of HCT116 and HeLa cells

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Supramolecule #1: gamma-tubulin ring complex in human centrosomes of HCT116 and HeL...

SupramoleculeName: gamma-tubulin ring complex in human centrosomes of HCT116 and HeLa cells
type: cell / ID: 1 / Parent: 0
Details: Data from HeLa cells were obtained from EMPIARC-200003 dataset of human centrosomes
Source (natural)Organism: Homo sapiens (human) / Strain: HCT116 & HeLa

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 3.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.4 µm / Nominal defocus min: 6.1000000000000005 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 30.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 562
ExtractionNumber tomograms: 14 / Number images used: 8260 / Software: (Name: PyTom (ver. 0.971), Warp (ver. 1.09))
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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