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- EMDB-26483: Actin organization during clathrin-mediated endocytosis -

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Basic information

Entry
Database: EMDB / ID: EMD-26483
TitleActin organization during clathrin-mediated endocytosis
Map dataActin organization during clathrin-mediated endocytosis
Sample
  • Cell: Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.
KeywordsClathrin / Actin / ENDOCYTOSIS
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsSerwas D / Akamatsu M / Moayed A / Vegesna K / Vasan R / Hill JM / Schoeneberg J / Davies KM / Rangamani P / Drubin DG
Funding support United States, France, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118149 United States
Human Frontier Science Program (HFSP)LT000234/2018-L France
CitationJournal: Dev Cell / Year: 2022
Title: Mechanistic insights into actin force generation during vesicle formation from cryo-electron tomography.
Authors: Daniel Serwas / Matthew Akamatsu / Amir Moayed / Karthik Vegesna / Ritvik Vasan / Jennifer M Hill / Johannes Schöneberg / Karen M Davies / Padmini Rangamani / David G Drubin /
Abstract: Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly ...Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization and that assembly is triggered from ∼4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.
History
DepositionMar 24, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26483.png

Downloads & links

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Map

FileDownload / File: emd_26483.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationActin organization during clathrin-mediated endocytosis
Voxel sizeX=Y=Z: 5.943 Å
Density
Minimum - Maximum-128.0 - 125.0
Average (Standard dev.)0.1997872 (±9.37914)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-205
Dimensions18551919410
Spacing19191855410
CellA: 11404.616 Å / B: 11024.265 Å / C: 2436.63 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.

EntireName: Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.
Components
  • Cell: Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.

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Supramolecule #1: Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.

SupramoleculeName: Clathrin-coated vesicle and associated actin network in SK-MEL-2 cell.
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: SK-MEL-2

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.07 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.0004994250000000001 µm / Nominal defocus min: -0.00162641 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 61

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