+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26484 | |||||||||
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Title | In situ map of the clathrin hub | |||||||||
Map data | In situ map of the clathrin hub | |||||||||
Sample |
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Keywords | Clathrin / Actin / ENDOCYTOSIS | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 27.0 Å | |||||||||
Authors | Serwas 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
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26484.map.gz | 1.8 MB | EMDB map data format | |
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Header (meta data) | emd-26484-v30.xml emd-26484.xml | 13.5 KB 13.5 KB | Display Display | EMDB header |
Images | emd_26484.png | 42.8 KB | ||
Filedesc metadata | emd-26484.cif.gz | 4.4 KB | ||
Others | emd_26484_half_map_1.map.gz emd_26484_half_map_2.map.gz | 469.9 KB 467.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26484 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26484 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_26484.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | In situ map of the clathrin hub | ||||||||||||||||||||
Voxel size | X=Y=Z: 5.943 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half Map 1
File | emd_26484_half_map_1.map | ||||||||||||
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Annotation | Half Map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 2
File | emd_26484_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : In situ map of the clathrin hub
Entire | Name: In situ map of the clathrin hub |
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Components |
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-Supramolecule #1: In situ map of the clathrin hub
Supramolecule | Name: In situ map of the clathrin hub / type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Homo sapiens (human) / Strain: SK-MEL-2 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 |
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Grid | Model: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.0152562 µm / Nominal defocus min: 0.0052084 µm |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 2.5 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Extraction | Number tomograms: 7 / Number images used: 561 |
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Final angle assignment | Type: NOT APPLICABLE |
Final reconstruction | Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software: (Name: IMOD, PEET) Details: Half maps were generated using the fnHalfVolume function of calcFSC in PEET rather than "gold standard" FSC. Number subtomograms used: 472 |