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- EMDB-11715: Cryo-EM reconstruction of the di-hexameric endocytic adaptor comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-11715
TitleCryo-EM reconstruction of the di-hexameric endocytic adaptor complex AENTH (ENTH F5A/L12A/V13A mutant)
Map datafinal, sharpened map
Sample
  • Complex: 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2
    • Protein or peptide: ANTH domain of Sla-2
    • Protein or peptide: ENTH domain of Epsin-1
Function / homology
Function and homology information


Cargo recognition for clathrin-mediated endocytosis / actin cortical patch assembly / clathrin vesicle coat / clathrin light chain binding / incipient cellular bud site / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / actin cortical patch / clathrin coat assembly / clathrin adaptor activity ...Cargo recognition for clathrin-mediated endocytosis / actin cortical patch assembly / clathrin vesicle coat / clathrin light chain binding / incipient cellular bud site / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / actin cortical patch / clathrin coat assembly / clathrin adaptor activity / cellular bud neck / mating projection tip / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / clathrin-coated vesicle / clathrin binding / K63-linked polyubiquitin modification-dependent protein binding / cortical actin cytoskeleton / ubiquitin binding / actin filament organization / phospholipid binding / endocytosis / actin filament binding / early endosome / endosome / plasma membrane / cytoplasm
Similarity search - Function
Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain ...Sla2 family / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / I/LWEQ domain / I/LWEQ domain superfamily / I/LWEQ domain / I/LWEQ domain profile. / I/LWEQ domain / ENTH/VHS / Ubiquitin-interacting motif. / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile.
Similarity search - Domain/homology
Protein SLA2 / Epsin-1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsKlebl DP / Lizarrondo J / Sobott F / Garcia-Alai MM / Muench SP
Funding support Germany, United Kingdom, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SK 305/1-1 Germany
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.
Authors: Javier Lizarrondo / David P Klebl / Stephan Niebling / Marc Abella / Martin A Schroer / Haydyn D T Mertens / Katharina Veith / Roland Thuenauer / Dmitri I Svergun / Michal Skruzny / Frank ...Authors: Javier Lizarrondo / David P Klebl / Stephan Niebling / Marc Abella / Martin A Schroer / Haydyn D T Mertens / Katharina Veith / Roland Thuenauer / Dmitri I Svergun / Michal Skruzny / Frank Sobott / Stephen P Muench / Maria M Garcia-Alai /
Abstract: During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat ...During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.
History
DepositionSep 10, 2020-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11715.png

Downloads & links

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Map

FileDownload / File: emd_11715.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal, sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.13 Å/pix.
x 128 pix.
= 272.64 Å		2.13 Å/pix.
x 128 pix.
= 272.64 Å		2.13 Å/pix.
x 128 pix.
= 272.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.13 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.27953807 - 0.5100174
Average (Standard dev.)0.0014697352 (±0.018684097)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 272.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.132.132.13
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z272.640272.640272.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.2800.5100.001

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Supplemental data

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Mask #1

Fileemd_11715_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11715_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11715_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2

EntireName: 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2
Components
  • Complex: 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2
    • Protein or peptide: ANTH domain of Sla-2
    • Protein or peptide: ENTH domain of Epsin-1

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Supramolecule #1: 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2

SupramoleculeName: 12 mer of 6 Sla2 ANTH and 6 Epsin-1 ENTH domains in complex with PIP2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: ANTH domain of Sla-2

MacromoleculeName: ANTH domain of Sla-2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGSMSRID SDLQKALKKA CSVEETAPKR KHVRACIVYT WDHQSSKAVF TTLKTLPLAN DEVQLFKMLI VLHKIIQEGH PSALAEAIRD RDWIRSLGRV HSGGSSYSKL IREYVRYLVL KLDFHAHHRG FNNGTFEYEE YVSLVSVSDP DEGYETILDL MSLQDSLDEF ...String:
GAMGSMSRID SDLQKALKKA CSVEETAPKR KHVRACIVYT WDHQSSKAVF TTLKTLPLAN DEVQLFKMLI VLHKIIQEGH PSALAEAIRD RDWIRSLGRV HSGGSSYSKL IREYVRYLVL KLDFHAHHRG FNNGTFEYEE YVSLVSVSDP DEGYETILDL MSLQDSLDEF SQIIFASIQS ERRNTECKIS ALIPLIAESY GIYKFITSML RAMHRQLNDA EGDAALQPLK ERYELQHARL FEFYADCSSV KYLTTLVTIP KLPVDAPDVF LINDVDESKE IKFKKREPSV T

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Macromolecule #2: ENTH domain of Epsin-1

MacromoleculeName: ENTH domain of Epsin-1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GAMGSMSKQA VRSAKNAAKG YSSTQVLVRN ATSNDNHQVS KDSLIELAEK SYDSADFFEI MDMLDKRLND KGKYWRHIAK ALTVIDYLIR FGSENCVLWC RENLYIIKTL KEFRHEDDEG IDQGQIVRVK AKELTALLSD DERLNEERNM NIKGRNRKGR RR

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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

Concentration0.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV
Details20 mM Tris, 250 mM NaCl and 1 mM DTT

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 72.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 142399
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 16206
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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