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- EMDB-14518: Chimaera of AP2 with FCHO2 linker domain, N1-N2 enriched population -

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Basic information

Entry
Database: EMDB / ID: EMD-14518
TitleChimaera of AP2 with FCHO2 linker domain, N1-N2 enriched population
Map dataN1-N2 enriched substructure of chimaera of AP2 with FCHO2 linker domain
Sample
  • Complex: AP2 core complex
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsKane Dickson V / Qu K / Owen DJ / Briggs JA / Zaccai NR
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: 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
DepositionMar 9, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateJun 1, 2022-
Current statusJun 1, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14518.png

Downloads & links

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Map

FileDownload / File: emd_14518.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationN1-N2 enriched substructure of chimaera of AP2 with FCHO2 linker domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 340 pix.
= 221.68 Å		0.65 Å/pix.
x 340 pix.
= 221.68 Å		0.65 Å/pix.
x 340 pix.
= 221.68 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.1902976 - 0.3196069
Average (Standard dev.)0.0011665495 (±0.009093331)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 221.68001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14518_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14518_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AP2 core complex

EntireName: AP2 core complex
Components
  • Complex: AP2 core complex

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Supramolecule #1: AP2 core complex

SupramoleculeName: 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)
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 203 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 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)
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2vgl

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 28392
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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