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

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Basic information

Entry
Database: PDB / ID: 5ahv
TitleCryo-EM structure of helical ANTH and ENTH tubules on PI(4,5)P2-containing membranes
Components
  • ANTH DOMAIN OF ENDOCYTIC ADAPTOR SLA2
  • ENTH DOMAIN OF EPSIN ENT1
KeywordsCLATHRIN-BINDING PROTEIN / CLATHRIN BINDING PROTEIN / EPSIN / HIP1R / ENTH / CLATHRIN ADAPTORS / ENDOCYTOSIS
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 / actin cortical patch / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / 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 / actin cortical patch / negative regulation of Arp2/3 complex-mediated actin nucleation / cellular bud tip / 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 / K63-linked polyubiquitin modification-dependent protein binding / clathrin 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 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsSkruzny, M. / Desfosses, A. / Prinz, S. / Dodonova, S.O. / Gieras, A. / Uetrecht, C. / Jakobi, A.J. / Abella, M. / Hagen, W.J.H. / Schulz, J. ...Skruzny, M. / Desfosses, A. / Prinz, S. / Dodonova, S.O. / Gieras, A. / Uetrecht, C. / Jakobi, A.J. / Abella, M. / Hagen, W.J.H. / Schulz, J. / Meijers, R. / Rybin, V. / Briggs, J.A.G. / 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, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 6, 2015Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 2.0Aug 9, 2017Group: Atomic model / Category: atom_site
Item: _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z
Revision 2.1Apr 20, 2022Group: Database references / Category: database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.2Mar 8, 2023Group: Derived calculations / Refinement description
Category: pdbx_initial_refinement_model / pdbx_struct_assembly_gen / pdbx_struct_oper_list
Item: _pdbx_struct_assembly_gen.oper_expression
Revision 2.3Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
E: ENTH DOMAIN OF EPSIN ENT1
F: ANTH DOMAIN OF ENDOCYTIC ADAPTOR SLA2


Theoretical massNumber of molelcules
Total (without water)48,7062
Polymers48,7062
Non-polymers00
Water00
1
E: ENTH DOMAIN OF EPSIN ENT1
F: ANTH DOMAIN OF ENDOCYTIC ADAPTOR SLA2
x 78


Theoretical massNumber of molelcules
Total (without water)3,799,102156
Polymers3,799,102156
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation77

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Components

#1: Protein ENTH DOMAIN OF EPSIN ENT1


Mass: 18005.471 Da / Num. of mol.: 1 / Fragment: RESIDUES 5-272
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PETM30 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q12518
#2: Protein ANTH DOMAIN OF ENDOCYTIC ADAPTOR SLA2 / TRANSMEMBRANE PROTEIN MOP2 / ANTH DOMAIN OF ENDOCYTIC ADAPTOR SLA2


Mass: 30700.961 Da / Num. of mol.: 1 / Fragment: RESIDUES 1-154
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PETM30 / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P33338

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: ANTH AND ENTH DOMAINS OF THE CLATHRIN ADAPTORS SLA2 AND ENT1, RESPECTIVELY
Type: ORGANELLE OR CELLULAR COMPONENT
Buffer solutionName: 20 MM HEPES, PH 7.5, 100 MM KCL / pH: 7.5 / Details: 20 MM HEPES, PH 7.5, 100 MM KCL
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 80, INSTRUMENT- HOMEMADE PLUNGER, METHOD- , APPLIED ON C-FLAT HOLEY CARBON COATED GRIDS (PROTOCHIPS) AND VITRIFIED,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Mar 1, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated magnification: 59000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 10 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 1419

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Processing

EM software
IDNameCategory
1DireXmodel fitting
2I-TASSERmodel fitting
3UCSF Chimeramodel fitting
4SPRING3D reconstruction
CTF correctionDetails: EACH PARTICLE
3D reconstructionResolution: 13.6 Å / Num. of particles: 457344 / Nominal pixel size: 1.78 Å / Actual pixel size: 1.78 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2896. (DEPOSITION ID: 13053).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--FLEXIBLE REFINEMENT PROTOCOL--HOMOLOGY MODEL
Atomic model buildingPDB-ID: 1H0A

1h0a


Accession code: 1H0A / Source name: PDB / Type: experimental model
RefinementHighest resolution: 13.6 Å
Refinement stepCycle: LAST / Highest resolution: 13.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3422 0 0 0 3422

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