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- PDB-9c5b: AP-3 bound to myristoylated Arf1 (Q71L) and LAMPI on a lipid nano... -

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Basic information

Entry
Database: PDB / ID: 9c5b
TitleAP-3 bound to myristoylated Arf1 (Q71L) and LAMPI on a lipid nanodisc; combined map
Components
  • (AP-3 complex subunit ...) x 4
  • ADP-ribosylation factor 1
  • Lysosome-associated membrane glycoprotein 1
KeywordsTRANSPORT PROTEIN / Adaptor Protein complex / AP-3 / Lysosomal transport / Endosomal transport / Protein trafficking
Function / homology
Function and homology information


synaptic vesicle coating / synaptic vesicle budding from endosome / establishment of protein localization to mitochondrial membrane involved in mitochondrial fission / clathrin-coated vesicle cargo loading, AP-3-mediated / skin epidermis development / AP-type membrane coat adaptor complex / synaptic vesicle membrane organization / regulation of organelle transport along microtubule / zinc ion import into lysosome / AP-3 adaptor complex ...synaptic vesicle coating / synaptic vesicle budding from endosome / establishment of protein localization to mitochondrial membrane involved in mitochondrial fission / clathrin-coated vesicle cargo loading, AP-3-mediated / skin epidermis development / AP-type membrane coat adaptor complex / synaptic vesicle membrane organization / regulation of organelle transport along microtubule / zinc ion import into lysosome / AP-3 adaptor complex / positive regulation of natural killer cell degranulation / neurotransmitter receptor transport, postsynaptic endosome to lysosome / anterograde synaptic vesicle transport / granzyme-mediated programmed cell death signaling pathway / phagolysosome membrane / microvesicle / Golgi to lysosome transport / endosome to melanosome transport / mitotic cleavage furrow ingression / trans-Golgi Network Vesicle Budding / Golgi to vacuole transport / establishment of protein localization to organelle / cytolytic granule membrane / synaptic vesicle recycling / postsynaptic recycling endosome / presynaptic endosome / clathrin adaptor complex / platelet dense granule organization / Glycosphingolipid transport / regulation of receptor internalization / melanosome assembly / granulocyte differentiation / Intra-Golgi traffic / regulation of Arp2/3 complex-mediated actin nucleation / postsynaptic neurotransmitter receptor internalization / GTP-dependent protein binding / positive regulation of NK T cell differentiation / Synthesis of PIPs at the Golgi membrane / clathrin-coated vesicle membrane / lysosomal lumen acidification / positive regulation of natural killer cell mediated cytotoxicity / antigen processing and presentation, exogenous lipid antigen via MHC class Ib / protein targeting to vacuole / protein targeting to lysosome / melanosome organization / respiratory system process / anterograde axonal transport / Nef Mediated CD4 Down-regulation / intracellular zinc ion homeostasis / dendritic spine organization / protein localization to membrane / protein localization to cell surface / long-term synaptic depression / azurophil granule membrane / lysosome organization / COPI-dependent Golgi-to-ER retrograde traffic / Lysosome Vesicle Biogenesis / toll-like receptor signaling pathway / ion channel inhibitor activity / Golgi Associated Vesicle Biogenesis / cell leading edge / lung morphogenesis / Association of TriC/CCT with target proteins during biosynthesis / Synthesis of PIPs at the plasma membrane / autolysosome / autophagosome membrane / ficolin-1-rich granule membrane / homeostasis of number of cells / intracellular copper ion homeostasis / single fertilization / intracellular transport / hematopoietic progenitor cell differentiation / COPI-mediated anterograde transport / transport vesicle / vesicle-mediated transport / axon cytoplasm / multivesicular body / MHC class II antigen presentation / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / cytoplasmic vesicle membrane / sarcomere / small monomeric GTPase / intracellular protein transport / mRNA transcription by RNA polymerase II / terminal bouton / cell morphogenesis / sarcolemma / protein modification process / small GTPase binding / cellular response to virus / endocytosis / blood coagulation / Signaling by BRAF and RAF1 fusions / late endosome membrane / late endosome / synaptic vesicle / insulin receptor signaling pathway / melanosome / presynapse / virus receptor activity
Similarity search - Function
AP-3 complex subunit sigma / AP-3 complex subunit delta domain, metazoa / AP-3 complex subunit delta-1 / 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 ...AP-3 complex subunit sigma / AP-3 complex subunit delta domain, metazoa / AP-3 complex subunit delta-1 / 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 / Lysosome-associated membrane glycoprotein, conserved site / AP-3 complex subunit beta / : / Lysosome-associated membrane glycoprotein 2, transmembrane segment / Lysosome-associated membrane glycoproteins duplicated domain signature. / LAMP glycoproteins transmembrane and cytoplasmic domain signature. / Lysosome-associated membrane glycoprotein / : / Lysosome-associated membrane glycoprotein 2-like, luminal domains / Lysosome-associated membrane glycoprotein family profile. / Adaptor protein complex, sigma subunit / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / ADP-ribosylation factor 1-5 / 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 complex, mu/sigma subunit / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Mu homology domain / Mu homology domain (MHD) profile. / Small GTPase superfamily, ARF type / small GTPase Arf family 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
GUANOSINE-5'-TRIPHOSPHATE / AP-3 complex subunit beta-1 / AP-3 complex subunit delta-1 / Lysosome-associated membrane glycoprotein 1 / ADP-ribosylation factor 1 / AP-3 complex subunit sigma-1 / AP-3 complex subunit mu-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
Model detailsMODEL GENERATED BY ROSETTA VERSION 2020.08+release.cb1caba
AuthorsBegley, M.C. / Baker, R.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM150960 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: A structure-based mechanism for initiation of AP-3 coated vesicle formation.
Authors: Matthew Begley / Mahira Aragon / Richard W Baker /
Abstract: Adaptor protein complex-3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 adopts a constitutively open conformation ...Adaptor protein complex-3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 adopts a constitutively open conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy. Similar to yeast AP-3, human AP-3 is in a constitutively open conformation. To reconstitute AP-3 activation by adenosine di-phosphate (ADP)-ribosylation factor 1 (Arf1), a small guanosine tri-phosphate (GTP)ase, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures showing the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane recruitment is driven by one of two predicted Arf1 binding sites, which flexibly tethers AP-3 to the membrane. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher-order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3-mediated vesicle coat assembly.
History
DepositionJun 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ADP-ribosylation factor 1
C: ADP-ribosylation factor 1
D: AP-3 complex subunit delta-1
M: AP-3 complex subunit mu-1
S: AP-3 complex subunit sigma-1
Y: Lysosome-associated membrane glycoprotein 1
B: AP-3 complex subunit beta-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,65111
Polymers257,5567
Non-polymers1,0954
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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AP-3 complex subunit ... , 4 types, 4 molecules DMSB

#2: Protein AP-3 complex subunit delta-1 / AP-3 complex subunit delta / Adaptor-related protein complex 3 subunit delta-1 / Delta-adaptin


Mass: 69381.977 Da / Num. of mol.: 1 / Fragment: residues 1-617
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3D1, PRO0039 / Production host: Escherichia coli (E. coli) / References: UniProt: O14617
#3: Protein AP-3 complex subunit mu-1 / AP-3 adaptor complex mu3A subunit / Adaptor-related protein complex 3 subunit mu-1 / Mu-adaptin 3A ...AP-3 adaptor complex mu3A subunit / Adaptor-related protein complex 3 subunit mu-1 / Mu-adaptin 3A / Mu3A-adaptin


Mass: 46989.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3M1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9Y2T2
#4: 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: 21755.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3S1, CLAPS3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q92572
#6: Protein AP-3 complex subunit beta-1 / Adaptor protein complex AP-3 subunit beta-1 / Adaptor-related protein complex 3 subunit beta-1 / ...Adaptor protein complex AP-3 subunit beta-1 / Adaptor-related protein complex 3 subunit beta-1 / Beta-3A-adaptin / Clathrin assembly protein complex 3 beta-1 large chain


Mass: 76499.609 Da / Num. of mol.: 1 / Fragment: residues 1-677
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3B1, ADTB3A / Production host: Escherichia coli (E. coli) / References: UniProt: O00203

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Protein / Protein/peptide , 2 types, 3 molecules ACY

#1: Protein ADP-ribosylation factor 1


Mass: 20775.812 Da / Num. of mol.: 2 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Details: Q to L mutation at position 71 / Source: (gene. exp.) Homo sapiens (human) / Gene: ARF1 / Production host: Escherichia coli (E. coli) / References: UniProt: P84077, small monomeric GTPase
#5: Protein/peptide Lysosome-associated membrane glycoprotein 1 / LAMP-1 / Lysosome-associated membrane protein 1 / CD107 antigen-like family member A


Mass: 1377.553 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Synthetic peptide with an oleic acid conjugation / Source: (synth.) Homo sapiens (human) / References: UniProt: P11279

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Non-polymers , 2 types, 4 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationN

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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: AP-3 bound to myristoylated Arf1 (Q71L) and LAMPI on a lipid nanodisc; combined map
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.215 MDa / Experimental value: NO
Buffer solutionpH: 7.4 / Details: 1x PBS (pH 7.4), 300mM NaCl, 1mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
12.7 mMPotassium ChlorideKCl1
210 mMSodium Phosphate (Dibasic)Na2HPO41
31.8 mMPotassium Phosphate (Monobasic)KH2PO41
4300 mMSodium ChlorideNaCl1
51 mMTCEPC9H15O6P1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Specimen appeared as a monodisperse peak via size exclusion chromatography (SEC)
Specimen supportDetails: Used Quantifoil Active grids - backside gold coated before plasma cleaning. 12 mA used for plasma cleaning.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296 K / Details: Commercialized version - chameleon

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1400 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53.4 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
Details: 2 datasets collected, processed independently, and merged.
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2Leginonimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraX1.7.1model fitting
9cryoSPARC4.4.1initial Euler assignment
10cryoSPARC4.4.1final Euler assignment
11cryoSPARC4.4.1classification
12cryoSPARC4.4.13D reconstruction
13Rosetta3.12model refinement
14PHENIX1.21.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Mixture of blob picker, template picker, crYOLO, and Topaz
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176749
Details: Non-Uniform refinement in cryoSPARC. Map is a composite map of two focused refinements, combined using phenix.combine_focused_maps.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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