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- PDB-9c5a: AP-3 Arf1 dimeric interface, focused refinement -

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Basic information

Entry
Database: PDB / ID: 9c5a
TitleAP-3 Arf1 dimeric interface, focused refinement
Components
  • (AP-3 complex subunit ...) x 2
  • ADP-ribosylation factor 1
  • Lysosome-associated membrane glycoprotein 1
KeywordsTRANSPORT PROTEIN / Adaptor Protein complex / Endosomal Trafficking / Lysosomal Trafficking / Protein transport / AP complex
Function / homology
Function and homology information


establishment of protein localization to mitochondrial membrane involved in mitochondrial fission / clathrin-coated vesicle cargo loading, AP-3-mediated / skin epidermis development / regulation of organelle transport along microtubule / AP-3 adaptor complex / positive regulation of natural killer cell degranulation / anterograde synaptic vesicle transport / granzyme-mediated programmed cell death signaling pathway / phagolysosome membrane / microvesicle ...establishment of protein localization to mitochondrial membrane involved in mitochondrial fission / clathrin-coated vesicle cargo loading, AP-3-mediated / skin epidermis development / regulation of organelle transport along microtubule / AP-3 adaptor complex / positive regulation of natural killer cell degranulation / anterograde synaptic vesicle transport / granzyme-mediated programmed cell death signaling pathway / phagolysosome membrane / microvesicle / Golgi to lysosome transport / establishment of protein localization to organelle / mitotic cleavage furrow ingression / cytolytic granule membrane / trans-Golgi Network Vesicle Budding / postsynaptic recycling endosome / clathrin adaptor complex / platelet dense granule organization / Glycosphingolipid transport / melanosome assembly / regulation of receptor internalization / Intra-Golgi traffic / regulation of Arp2/3 complex-mediated actin nucleation / postsynaptic neurotransmitter receptor internalization / granulocyte differentiation / 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 / GTP-dependent protein binding / protein targeting to lysosome / melanosome organization / respiratory system process / anterograde axonal transport / Nef Mediated CD4 Down-regulation / intracellular zinc ion homeostasis / dendritic spine organization / long-term synaptic depression / protein localization to cell surface / COPI-dependent Golgi-to-ER retrograde traffic / azurophil granule membrane / lysosome organization / toll-like receptor signaling pathway / Lysosome Vesicle Biogenesis / ion channel inhibitor activity / Golgi Associated Vesicle Biogenesis / lung morphogenesis / Association of TriC/CCT with target proteins during biosynthesis / Synthesis of PIPs at the plasma membrane / cell leading edge / autolysosome / autophagosome membrane / homeostasis of number of cells / ficolin-1-rich granule membrane / intracellular copper ion homeostasis / single fertilization / intracellular transport / hematopoietic progenitor cell differentiation / COPI-mediated anterograde transport / vesicle-mediated transport / multivesicular body / axon cytoplasm / 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 / sarcolemma / cellular response to virus / protein modification process / small GTPase binding / endocytosis / cell morphogenesis / blood coagulation / synaptic vesicle / late endosome membrane / Signaling by BRAF and RAF1 fusions / melanosome / late endosome / virus receptor activity / cytoplasmic vesicle / protein phosphatase binding / spermatogenesis / early endosome / lysosome / neuron projection / endosome membrane / postsynaptic density / protein stabilization / inflammatory response / Golgi membrane / protein domain specific binding / lysosomal membrane / external side of plasma membrane / focal adhesion / GTPase activity
Similarity search - Function
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 / Lysosome-associated membrane glycoprotein, conserved site / AP-3 complex subunit beta / : ...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 / 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. / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / ADP-ribosylation factor 1-5 / 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 / Adaptin N terminal region / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Small GTPase superfamily, ARF type / 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
GUANOSINE-5'-TRIPHOSPHATE / AP-3 complex subunit beta-1 / Lysosome-associated membrane glycoprotein 1 / ADP-ribosylation factor 1 / AP-3 complex subunit mu-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
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
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Revision 1.1Mar 5, 2025Group: Data collection / Database references / 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
B: AP-3 complex subunit beta-1
M: AP-3 complex subunit mu-1
C: ADP-ribosylation factor 1
Y: Lysosome-associated membrane glycoprotein 1
b: AP-3 complex subunit beta-1
m: AP-3 complex subunit mu-1
c: ADP-ribosylation factor 1
y: Lysosome-associated membrane glycoprotein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)293,95512
Polymers292,8608
Non-polymers1,0954
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

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AP-3 complex subunit ... , 2 types, 4 molecules BbMm

#1: 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: 77286.523 Da / Num. of mol.: 2 / 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) / Strain (production host): BL21 Star / References: UniProt: O00203
#2: 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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP3M1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Star / References: UniProt: Q9Y2T2

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Protein / Protein/peptide , 2 types, 4 molecules CcYy

#3: Protein ADP-ribosylation factor 1


Mass: 20775.812 Da / Num. of mol.: 2 / Mutation: Q71L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARF1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P84077, small monomeric GTPase
#4: 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.: 2 / Fragment: C-terminal cytoplasmic tail / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P11279

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: 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 Arf1 dimeric interface, focused refinement / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.256 MDa / Experimental value: YES
Buffer solutionpH: 7.4
Details: 1x PBS (pH 7.4), 150mM NaCl, 1mM TCEP, 2mM GTP, 5mM EDTA, 10mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
12.7 mMPotassium ChlorideKCl1
210 mMSodium Phosphate (Dibasic)Na2HPO41
31.8 mMPotassium Phosphate (Monobasic)KH2PO41
4150 mMSodium ChlorideNaCl1
51 mMTCEPC9H15O6P1
65 mMEDTAC10H16N2O81
710 mMMagnesium ChlorideMgCl21
82 mMGTPC10H16N5O14P31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: AP-3 dimer bound to myristoylated Arf1 (Q71L) and LAMP1 on a lipid nanodisc; focused refinement on Arf1 dimeric interface
Specimen supportDetails: Quantifoil Active grids (SPT Labtech) with backside gold coated. Plasma was 12mA for 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: 294 K / Details: Commercial form of the chameleon (SPT Labtech)

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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 magnification: 81000 X / Nominal defocus max: 1400 nm / Nominal defocus min: 400 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53.37 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
Details: 2 datasets collected independently and merged for processing

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2Leginonimage acquisition
4cryoSPARC4.4.1CTF correction
7UCSF ChimeraXmodel 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
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122155 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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