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- PDB-7p3z: Homology model of the full-length AP-3 complex in a stretched ope... -

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Basic information

Entry
Database: PDB / ID: 7p3z
TitleHomology model of the full-length AP-3 complex in a stretched open conformation
Components
  • AP complex subunit sigma
  • AP-3 complex subunit delta
  • AP-3 complex subunit mu
  • Y55_G0035830.mRNA.1.CDS.1
KeywordsTRANSPORT PROTEIN / adaptor protein / vesicle transport / AP-3 / homology model
Function / homology
Function and homology information


AP-3 adaptor complex / clathrin adaptor complex / Golgi to vacuole transport / protein targeting to vacuole / membrane coat / vesicle-mediated transport / intracellular protein transport / cytoplasmic vesicle membrane / endocytosis / cytoplasmic vesicle / Golgi apparatus
Similarity search - Function
AP-3 complex subunit beta / Adaptor protein complex AP-3, delta subunit / Adaptor protein complex, sigma subunit / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. ...AP-3 complex subunit beta / Adaptor protein complex AP-3, delta subunit / Adaptor protein complex, sigma subunit / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / : / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Longin-like domain superfamily / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
AP complex subunit sigma / Y55_G0035830.mRNA.1.CDS.1 / AP-3 complex subunit delta / AP-3 complex subunit mu
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10.5 Å
AuthorsSchubert, E. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)RA1781/2-4 Germany
CitationJournal: J Biol Chem / Year: 2021
Title: Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.
Authors: Jannis Schoppe / Evelyn Schubert / Amir Apelbaum / Erdal Yavavli / Oliver Birkholz / Heike Stephanowitz / Yaping Han / Angela Perz / Oliver Hofnagel / Fan Liu / Jacob Piehler / Stefan ...Authors: Jannis Schoppe / Evelyn Schubert / Amir Apelbaum / Erdal Yavavli / Oliver Birkholz / Heike Stephanowitz / Yaping Han / Angela Perz / Oliver Hofnagel / Fan Liu / Jacob Piehler / Stefan Raunser / Christian Ungermann /
Abstract: Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane ...Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.
History
DepositionJul 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 29, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.year / _em_admin.last_update
Revision 1.2Nov 17, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.3Dec 8, 2021Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

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Assembly

Deposited unit
A: AP-3 complex subunit delta
B: Y55_G0035830.mRNA.1.CDS.1
M: AP-3 complex subunit mu
S: AP complex subunit sigma


Theoretical massNumber of molelcules
Total (without water)279,4664
Polymers279,4664
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein AP-3 complex subunit delta


Mass: 110903.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS5975 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: A0A7I9C4X2
#2: Protein Y55_G0035830.mRNA.1.CDS.1


Mass: 91712.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS2762 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: A0A7I9BYB9
#3: Protein AP-3 complex subunit mu / AP-3 adaptor complex mu3A subunit / Adaptor-related protein complex 3 subunit mu / Mu-adaptin 3A / Mu3-adaptin


Mass: 54899.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: P38153
#4: Protein AP complex subunit sigma


Mass: 21951.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS3239, PACBIOSEQ_LOCUS3310, SCNYR20_0009016500, SCP684_0009016000
Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: A0A6L1B7P9

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Full-length AP-3 complex from Saccharomyces cerevisiae
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Buffer solutionpH: 7.4 / Details: 20 mM Hepes pH 7.4 150 mM NaCl 1.5 mM MgCl2
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: The sample was blotted using a 2.5 s blotting time and 0 blotting force with 100% humidity at 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 1500 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 81 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
4SPHIRE1.3CTF correction
7UCSF Chimera1.13.1model fittingRigid body Fitting (FitToMap)
8iMODFITv1.51model fittingFlexible fitting of homology models
11SPHIRE1.3initial Euler assignment
12SPHIRE1.3final Euler assignmentMERIDIEN
14SPHIRE1.33D reconstructionMERIDIEN
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 958892
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 10.5 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 20312 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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