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Yorodumi- EMDB-14518: Chimaera of AP2 with FCHO2 linker domain, N1-N2 enriched population -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14518 | |||||||||
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Title | Chimaera of AP2 with FCHO2 linker domain, N1-N2 enriched population | |||||||||
Map data | N1-N2 enriched substructure of chimaera of AP2 with FCHO2 linker domain | |||||||||
Sample |
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Biological species | Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.7 Å | |||||||||
Authors | Kane Dickson V / Qu K / Owen DJ / Briggs JA / Zaccai NR | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: Sci Adv / Year: 2022 Title: FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch. Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / ...Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / Sally R Gray / Kun Qu / Philip R Evans / Marco Fritzsche / Filip Sroubek / Stefan Höning / John A G Briggs / Bernard T Kelly / David J Owen / Linton M Traub / Abstract: Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane- ...Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14518.map.gz | 11.4 MB | EMDB map data format | |
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Header (meta data) | emd-14518-v30.xml emd-14518.xml | 14.8 KB 14.8 KB | Display Display | EMDB header |
Images | emd_14518.png | 83.6 KB | ||
Others | emd_14518_half_map_1.map.gz emd_14518_half_map_2.map.gz | 139.3 MB 139.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14518 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14518 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_14518.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | N1-N2 enriched substructure of chimaera of AP2 with FCHO2 linker domain | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.652 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_14518_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_14518_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AP2 core complex
Entire | Name: AP2 core complex |
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Components |
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-Supramolecule #1: AP2 core complex
Supramolecule | Name: AP2 core complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 Details: Ternary complex of AP2 core expressed as part of a chimaera with FCHO2 linker (not modelled) |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Molecular weight | Theoretical: 203 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.4 mg/mL |
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Buffer | pH: 7.6 Details: 50% HKT buffer (10mM Hepes, 10mM Tris 120mM potassium acetate pH 7.2) and 50% Core buffer (10mM Tris, 250mM NaCl, pH 8) |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
Details | This sample was monodisperse. |
-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: 2.9 µm / Nominal defocus min: 0.9 µm |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 28392 |