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- EMDB-46973: Plasma membrane bound clathrin vertex (HEK293) -

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Basic information

Entry
Database: EMDB / ID: EMD-46973
TitlePlasma membrane bound clathrin vertex (HEK293)
Map data
Sample
  • Complex: clathrin
Keywordsclathrin / membrane / lattice / complex / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 19.8 Å
AuthorsMichalak DJ / Sochacki KA / Taraska JW
Funding support United States, 1 items
OrganizationGrant numberCountry
Chan Zuckerberg Initiative United States
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-electron tomography pipeline for plasma membranes.
Authors: Willy W Sun / Dennis J Michalak / Kem A Sochacki / Prasanthi Kunamaneni / Marco A Alfonzo-Méndez / Andreas M Arnold / Marie-Paule Strub / Jenny E Hinshaw / Justin W Taraska /
Abstract: Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane ...Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells.
History
DepositionSep 10, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46973.png

Downloads & links

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Map

FileDownload / File: emd_46973.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 256 pix.
= 541.696 Å		2.12 Å/pix.
x 256 pix.
= 541.696 Å		2.12 Å/pix.
x 256 pix.
= 541.696 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.116 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.33176085 - 0.9418986
Average (Standard dev.)0.003495048 (±0.059915897)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 541.696 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_46973_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_46973_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 : clathrin

EntireName: clathrin
Components
  • Complex: clathrin

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Supramolecule #1: clathrin

SupramoleculeName: clathrin / type: complex / 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 statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE
DetailsUnroofed apical plasma membrane

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 19.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 1550
ExtractionNumber tomograms: 20 / Number images used: 2435 / Software - Name: RELION (ver. 5)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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