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- EMDB-2896: Cryo-EM structure of helical ANTH and ENTH tubules on PI(4,5)P2-c... -

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Basic information

Entry
Database: EMDB / ID: EMD-2896
TitleCryo-EM structure of helical ANTH and ENTH tubules on PI(4,5)P2-containing membranes
Map dataReconstruction of the 10.04 units per turn, 127.2 Angstrom pitch, helical assembly of ANTH-ENTH on PIP2 containing membrane
Sample
  • Sample: ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, respectively.
  • Protein or peptide: ANTH domain of endocytic adaptor Sla2
  • Protein or peptide: ENTH domain of epsin Ent1
Keywordsepsin / Hip1R / ENTH / ANTH / clathrin adaptors / endocytosis / PI(4 / 5)P2
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 / clathrin coat assembly / cellular bud tip / actin cortical patch / 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 / clathrin coat assembly / cellular bud tip / actin cortical patch / 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 / actin filament organization / ubiquitin binding / phospholipid binding / endocytosis / actin filament binding / early endosome / endosome / plasma membrane / cytoplasm
Similarity search - Function
Sla2 family / AP180 N-terminal homology (ANTH) domain / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / ENTH domain / I/LWEQ domain ...Sla2 family / AP180 N-terminal homology (ANTH) domain / AP180 N-terminal homology (ANTH) domain / ANTH domain / ENTH domain / Epsin N-terminal homology (ENTH) domain / ENTH domain profile. / ENTH domain / 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: 13.6 Å
AuthorsSkruzny M / Desfosses A / Prinz S / Dodonova SO / Gieras A / Uetrecht C / Jakobi AJ / Abella M / Hagen WJH / Schulz J ...Skruzny M / Desfosses A / Prinz S / Dodonova SO / Gieras A / Uetrecht C / Jakobi AJ / Abella M / Hagen WJH / Schulz J / Meijers R / Rybin V / Briggs JAG / Sachse C / Kaksonen M
CitationJournal: Dev Cell / Year: 2015
Title: An organized co-assembly of clathrin adaptors is essential for endocytosis.
Authors: Michal Skruzny / Ambroise Desfosses / Simone Prinz / Svetlana O Dodonova / Anna Gieras / Charlotte Uetrecht / Arjen J Jakobi / Marc Abella / Wim J H Hagen / Joachim Schulz / Rob Meijers / ...Authors: Michal Skruzny / Ambroise Desfosses / Simone Prinz / Svetlana O Dodonova / Anna Gieras / Charlotte Uetrecht / Arjen J Jakobi / Marc Abella / Wim J H Hagen / Joachim Schulz / Rob Meijers / Vladimir Rybin / John A G Briggs / Carsten Sachse / Marko Kaksonen /
Abstract: Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor ...Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process. Here we show that the membrane-binding domains of two conserved clathrin adaptors, Sla2 and Ent1, co-assemble in a PI(4,5)P2-dependent manner to form organized lattices on membranes. We determined the structure of the co-assembled lattice by electron cryo-microscopy and designed mutations that specifically impair the lattice formation in vitro. We show that these mutations block endocytosis in vivo. We suggest that clathrin adaptors not only link the polymerized clathrin to the membrane but also form an oligomeric structure, which is essential for membrane remodeling during endocytosis.
History
DepositionFeb 10, 2015-
Header (metadata) releaseMar 4, 2015-
Map releaseMay 6, 2015-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ahv
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ahv
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5ahv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2896_pre...

Downloads & links

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Map

FileDownload / File: emd_2896.map.gz / Format: CCP4 / Size: 73 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the 10.04 units per turn, 127.2 Angstrom pitch, helical assembly of ANTH-ENTH on PIP2 containing membrane
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.78 Å/pix.
x 340 pix.
= 605.2 Å		1.78 Å/pix.
x 240 pix.
= 427.2 Å		1.78 Å/pix.
x 240 pix.
= 427.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.78 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35 / Movie #1: 0.35
Minimum - Maximum-1.46910262 - 2.24971223
Average (Standard dev.)0.04299217 (±0.5508346)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-170
Dimensions240240340
Spacing240240340
CellA: 427.19998 Å / B: 427.19998 Å / C: 605.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z240240340
origin x/y/z0.0000.0000.000
length x/y/z427.200427.200605.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-120-120-170
NC/NR/NS240240340
D min/max/mean-1.4692.2500.043

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Supplemental data

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Sample components

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Entire : ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, res...

EntireName: ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, respectively.
Components
  • Sample: ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, respectively.
  • Protein or peptide: ANTH domain of endocytic adaptor Sla2
  • Protein or peptide: ENTH domain of epsin Ent1

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Supramolecule #1000: ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, res...

SupramoleculeName: ANTH and ENTH domains of the clathrin adaptors Sla2 and Ent1, respectively.
type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: ANTH domain of endocytic adaptor Sla2

MacromoleculeName: ANTH domain of endocytic adaptor Sla2 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: bakers yeast / Location in cell: plasma membrane
Molecular weightExperimental: 33.215 KDa / Theoretical: 33.21 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: CodonPlus / Recombinant plasmid: pETM30
SequenceUniProtKB: Protein SLA2 / InterPro: AP180 N-terminal homology (ANTH) domain

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Macromolecule #2: ENTH domain of epsin Ent1

MacromoleculeName: ENTH domain of epsin Ent1 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: bakers yeast / Location in cell: plasma membrane
Molecular weightExperimental: 18.87 KDa / Theoretical: 18.847 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: CodonPlus / Recombinant plasmid: pETM30
SequenceUniProtKB: Epsin-1 / InterPro: ENTH domain

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.5 / Details: 20 mM HEPES, pH 7.5; 100 mM KCl
GridDetails: on C-flat holey carbon coated grids (Protochips)
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER
Method: , applied on C-flat holey carbon coated grids (Protochips) and vitrified

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateMar 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1419 / Average electron dose: 10 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsProcessing done with SPRING
CTF correctionDetails: Each Particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 12.67 Å
Applied symmetry - Helical parameters - Δ&Phi: 35.857 °
Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 13.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPRING / Number images used: 457344

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Atomic model buiding 1

Initial modelPDB ID:

1h0a

SoftwareName: I-Tasser, Chimera, DireX
Detailshomology models were obtained by I-Tasser. Rigid-body docking was done in Chimera followed by flexible fitting done with DireX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5ahv:
Cryo-EM structure of helical ANTH and ENTH tubules on PI(4,5)P2-containing membranes

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Atomic model buiding 2

Initial modelPDB ID:

1hf8

SoftwareName: I-Tasser, Chimera, DireX
Detailshomology models were obtained by I-Tasser. Rigid-body docking was done in Chimera followed by flexible fitting done with DireX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5ahv:
Cryo-EM structure of helical ANTH and ENTH tubules on PI(4,5)P2-containing membranes

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