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- PDB-9rtw: Mammalian AP3 complex on tubular membranes (AP3 centered) -

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Basic information

Entry
Database: PDB / ID: 9rtw
TitleMammalian AP3 complex on tubular membranes (AP3 centered)
Components
  • ADP-ribosylation factor 1
  • AP-3 complex subunit beta
  • AP-3 complex subunit delta-1
  • AP-3 complex subunit mu-1
  • AP-3 complex subunit sigma-1
KeywordsENDOCYTOSIS / Cargo adapter / Coat / trafficking
Function / homology
Function and homology information


synaptic vesicle coating / synaptic vesicle membrane organization / clathrin-coated vesicle cargo loading, AP-3-mediated / zinc ion import into lysosome / AP-type membrane coat adaptor complex / AP-3 adaptor complex / neurotransmitter receptor transport, postsynaptic endosome to lysosome / anterograde synaptic vesicle transport / synaptic vesicle budding from endosome / endosome to melanosome transport ...synaptic vesicle coating / synaptic vesicle membrane organization / clathrin-coated vesicle cargo loading, AP-3-mediated / zinc ion import into lysosome / AP-type membrane coat adaptor complex / AP-3 adaptor complex / neurotransmitter receptor transport, postsynaptic endosome to lysosome / anterograde synaptic vesicle transport / synaptic vesicle budding from endosome / endosome to melanosome transport / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / platelet dense granule organization / synaptic vesicle recycling / melanosome assembly / Glycosphingolipid transport / clathrin adaptor complex / presynaptic endosome / regulation of receptor internalization / Intra-Golgi traffic / Golgi to vacuole transport / positive regulation of NK T cell differentiation / regulation of Arp2/3 complex-mediated actin nucleation / Synthesis of PIPs at the Golgi membrane / clathrin-coated vesicle membrane / antigen processing and presentation, exogenous lipid antigen via MHC class Ib / protein targeting to vacuole / melanosome organization / anterograde axonal transport / Nef Mediated CD4 Down-regulation / dendritic spine organization / protein localization to membrane / long-term synaptic depression / COPI-dependent Golgi-to-ER retrograde traffic / Lysosome Vesicle Biogenesis / Golgi Associated Vesicle Biogenesis / Synthesis of PIPs at the plasma membrane / cell leading edge / intracellular copper ion homeostasis / transport vesicle / COPI-mediated anterograde transport / vesicle-mediated transport / axon cytoplasm / MHC class II antigen presentation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / cytoplasmic vesicle membrane / small monomeric GTPase / sarcomere / intracellular protein transport / cellular response to virus / terminal bouton / insulin receptor signaling pathway / presynapse / G protein activity / early endosome / endosome membrane / neuron projection / postsynaptic density / postsynapse / Golgi membrane / protein domain specific binding / lysosomal membrane / focal adhesion / GTPase activity / GTP binding / glutamatergic synapse / magnesium ion binding / Golgi apparatus / positive regulation of transcription by RNA polymerase II / protein-containing complex / RNA binding / extracellular exosome / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
AP-3 complex subunit sigma / AP-3 complex subunit delta domain, metazoa / : / AP-3 complex subunit delta-1 / AP3 Mu C-terminal domain / Bovine leukaemia virus receptor (BLVR) / AP-3 complex subunit beta, C-terminal domain / AP-3 complex subunit beta 1, serine-rich domain / : / Clathrin-adaptor complex-3 beta-1 subunit C-terminal ...AP-3 complex subunit sigma / AP-3 complex subunit delta domain, metazoa / : / AP-3 complex subunit delta-1 / AP3 Mu C-terminal domain / Bovine leukaemia virus receptor (BLVR) / AP-3 complex subunit beta, C-terminal domain / AP-3 complex subunit beta 1, serine-rich domain / : / Clathrin-adaptor complex-3 beta-1 subunit C-terminal / Serine-rich region of AP3B1, clathrin-adaptor complex / AP-3 complex subunit beta-1, C-terminal domain / Clathrin-adaptor complex-3 beta-1 subunit C-terminal / Adaptor protein complex AP-3, delta subunit / AP-3 complex subunit beta / Adaptor protein complex, sigma subunit / ADP-ribosylation factor 1-5 / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / AP complex subunit beta / 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 complex, mu/sigma subunit / Adaptor complexes medium subunit family / Clathrin adaptor complex small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / AP-2 complex subunit mu, C-terminal superfamily / Adaptin N terminal region / Small GTPase superfamily, ARF type / Mu homology domain / Mu homology domain (MHD) profile. / Small GTPase Arf domain profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / Longin-like domain superfamily / ARF-like small GTPases; ARF, ADP-ribosylation factor / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
AP-3 complex subunit beta / Adaptor-related protein complex 3, mu 1 subunit / AP-3 complex subunit delta-1 / ADP-ribosylation factor 1 / AP-3 complex subunit sigma-1
Similarity search - Component
Biological speciesPan troglodytes (chimpanzee)
Homo sapiens (human)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.4 Å
AuthorsKaufman, J.G.G. / Tagiltsev, G. / Briggs, J.A.G. / Owen, D.J.
Funding support United Kingdom, Germany, European Union, 9items
OrganizationGrant numberCountry
Wellcome Trust227915/Z/23/Z United Kingdom
Wellcome Trust207455/Z/17/Z United Kingdom
Max Planck Society Germany
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N013433/1 United Kingdom
European Molecular Biology Organization (EMBO)ALTF-383-2022European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W005905/ United Kingdom
UK Research and Innovation (UKRI)UKRI715 United Kingdom
Wellcome Trust210481 United Kingdom
CitationJournal: Sci Adv / Year: 2026
Title: Architecture of clathrin-independent AP3:ARF1-coated carriers.
Authors: Jonathan G G Kaufman / Grigory Tagiltsev / Danièle S Stalder / Rebecca J Taylor / Ioana Sava / Hui Guo / Katarzyna A Ciazynska / Nathan R Zaccai / Sally R Gray / Yvonne Vallis / Stefan ...Authors: Jonathan G G Kaufman / Grigory Tagiltsev / Danièle S Stalder / Rebecca J Taylor / Ioana Sava / Hui Guo / Katarzyna A Ciazynska / Nathan R Zaccai / Sally R Gray / Yvonne Vallis / Stefan Höning / Bernard T Kelly / David C Gershlick / John A G Briggs / David J Owen /
Abstract: The AP3 complex mediates cargo sorting and carrier assembly for the trafficking of transmembrane proteins from endosomes to lysosomes. AP3 is generally believed to localize to clathrin-free, ARF1- ...The AP3 complex mediates cargo sorting and carrier assembly for the trafficking of transmembrane proteins from endosomes to lysosomes. AP3 is generally believed to localize to clathrin-free, ARF1-positive, elongated carriers in cells, but the architecture of AP3-based coats was unknown. Using in vitro reconstitution and cryo-electron tomography, we demonstrate that AP3:ARF1 spontaneously remodels membranes containing cargo and the phosphoinositide PI(3,5)P into tubular structures coated in spiraling rows of AP3 arches and ARF1 dimers. Targeted point mutations disrupting critical AP3:ARF1 and AP3:AP3 lattice interfaces disrupt AP3 recruitment, carrier formation, and lysosomal cargo trafficking in cells. We propose that AP3 generates tubular carriers on endosomes by organizing ARF1 dimers into elongated membrane-deforming arrays while simultaneously selecting cargo. By demonstrating that AP3:ARF1 can generate carriers without using a clathrin lattice, we explain the clathrin independence of AP3-mediated trafficking.
History
DepositionJul 3, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: AP-3 complex subunit beta
D: AP-3 complex subunit delta-1
E: ADP-ribosylation factor 1
F: ADP-ribosylation factor 1
G: ADP-ribosylation factor 1
H: ADP-ribosylation factor 1
M: AP-3 complex subunit mu-1
S: AP-3 complex subunit sigma-1


Theoretical massNumber of molelcules
Total (without water)274,4428
Polymers274,4428
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein AP-3 complex subunit beta


Mass: 63956.988 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pan troglodytes (chimpanzee) / Gene: AP3B1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A2I3SW12
#2: Protein AP-3 complex subunit delta-1 / AP-3 complex subunit delta / Adaptor-related protein complex 3 subunit delta-1 / Delta-adaptin


Mass: 69061.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3D1, PRO0039 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O14617
#3: Protein
ADP-ribosylation factor 1


Mass: 19209.930 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARF1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84077, small monomeric GTPase
#4: Protein AP-3 complex subunit mu-1


Mass: 47017.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap3m1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A6KKR5
#5: Protein AP-3 complex subunit sigma-1 / AP-3 complex subunit sigma-3A / Adaptor-related protein complex 3 subunit sigma-1 / Clathrin- ...AP-3 complex subunit sigma-3A / Adaptor-related protein complex 3 subunit sigma-1 / Clathrin-associated/assembly/adaptor protein / small 3 / Sigma-3A-adaptin / Sigma3A-adaptin / Sigma-adaptin 3a


Mass: 17566.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3S1, CLAPS3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q92572
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Mammalian AP-3 complex assembled on tubular membranes / Type: COMPLEX / Details: helical / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1592.786 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
220 mMTris(hydroxymethyl)aminomethane-HClTris-HCl1
31 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified recombinant AP3 complex was reconstituted with mystoylated ARF1 GTPase on GUV containing lipidated cargo motif sequences (TGN38:CKVTRRPKASDYQRL and MFSD12:GEHTPLLAPATC). ...Details: Purified recombinant AP3 complex was reconstituted with mystoylated ARF1 GTPase on GUV containing lipidated cargo motif sequences (TGN38:CKVTRRPKASDYQRL and MFSD12:GEHTPLLAPATC). Reconstituted AP3 spontaneously assembled to tubulate membranes.
Specimen supportGrid material: COPPER / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 284 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 6000 nm / Nominal defocus min: 3000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 3 e/Å2 / Avg electron dose per subtomogram: 120 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of real images: 1
EM imaging opticsEnergyfilter name: TFS Selectris X

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Processing

EM software
IDNameCategory
1UCSF Chimeravolume selection
2subTOMvolume selection
3Warpvolume selection
4Mvolume selection
5RELIONvolume selection
8WarpCTF correction
11UCSF ChimeraXmodel fitting
14RELIONfinal Euler assignment
15RELIONclassification
16RELION3D reconstruction
23Cootmodel refinement
CTF correctionDetails: 3DCTF in WARP / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42469 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 32 / Num. of volumes extracted: 121939 / Reference model: reference free STA
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Alphafold 3 (AF3), was used to generate 5 models of mammalian AP3 in complex with two copies of ARF1 in the GTP bound conformation. As the structure from STA was in a much more extended ...Details: Alphafold 3 (AF3), was used to generate 5 models of mammalian AP3 in complex with two copies of ARF1 in the GTP bound conformation. As the structure from STA was in a much more extended conformation than AF3 models or previous structures, direct rigid body docking of the complete AF3 model into the STA density was not possible. To create the C1 model the predicted complex was separated into three submodels, each of which was independently fitted into the map as a rigid body. Submodel 1 consisted of sigma3 (1-193), delta (1-387), delta-ARF1 (1-181). Submodel 2 consisted of mu3 (1-418), beta3 (1-472), beta3-ARF1. Submodel 3 consisted of beta3 (473-636) and delta (388-638) formed submodel 3. Rigid body fitting was performed using ChimeraX. This positioned truncated regions of submodels 1 and 2 in close proximity to their adjoining regions in submodel3, thus the split beta3 and delta chains were rejoined into single chains before real-space refinement in COOT to correct bond angles, distances and torsions. Using the previously resolved ARF1 dimer (PDBID: 9C5A), ARF1 was first superimposed with the existing ARF1 on beta3 and delta before rigid docking into the EM density. Flexible regions not accounted for sufficiently by modellable density were removed, these regions are listed as follows; beta3 (1-42), beta3 (259-292), delta (1-17), sigma3 (154-193) and ARF1 (1-13). Side chains and rotamers were not modelled and were therefore removed in ChimeraX.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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