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- EMDB-13187: Homology model of the full-length AP-3 complex in a compact open ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13187
TitleHomology model of the full-length AP-3 complex in a compact open conformation
Map data
Sample
  • Complex: Full-length AP-3 complex from Saccharomyces cerevisiae
    • Protein or peptide: AP-3 complex subunit delta
    • Protein or peptide: Y55_G0035830.mRNA.1.CDS.1
    • Protein or peptide: AP complex subunit sigma
    • Protein or peptide: AP-3 complex subunit mu
Keywordsadaptor protein / vesicle transport / AP-3 / homology model / TRANSPORT PROTEIN
Function / homology
Function and homology information


AP-3 adaptor complex / Golgi to vacuole transport / clathrin adaptor complex / membrane coat / protein targeting to vacuole / vesicle-mediated transport / cytoplasmic vesicle membrane / intracellular protein transport / endocytosis / cytoplasmic vesicle / Golgi apparatus
Similarity search - Function
Adaptor protein complex AP-3, delta subunit / AP-3 complex subunit beta / 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 protein complex AP-3, delta subunit / AP-3 complex subunit beta / 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 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. / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsSchubert E / Raunser S
Funding support Germany, 1 items
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, 2021-
Header (metadata) releaseSep 29, 2021-
Map releaseSep 29, 2021-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p3x
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13187.png

Downloads & links

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Map

FileDownload / File: emd_13187.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 264 pix.
= 282.48 Å		1.07 Å/pix.
x 264 pix.
= 282.48 Å		1.07 Å/pix.
x 264 pix.
= 282.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.014375185 - 0.049294703
Average (Standard dev.)0.000045470068 (±0.0021507565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 282.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z282.480282.480282.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.0140.0490.000

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Supplemental data

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Sample components

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Entire : Full-length AP-3 complex from Saccharomyces cerevisiae

EntireName: Full-length AP-3 complex from Saccharomyces cerevisiae
Components
  • Complex: Full-length AP-3 complex from Saccharomyces cerevisiae
    • Protein or peptide: AP-3 complex subunit delta
    • Protein or peptide: Y55_G0035830.mRNA.1.CDS.1
    • Protein or peptide: AP complex subunit sigma
    • Protein or peptide: AP-3 complex subunit mu

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Supramolecule #1: Full-length AP-3 complex from Saccharomyces cerevisiae

SupramoleculeName: Full-length AP-3 complex from Saccharomyces cerevisiae
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c

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Macromolecule #1: AP-3 complex subunit delta

MacromoleculeName: AP-3 complex subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 110.903016 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MTSLYAPGAE DIRQRLRPFG FFFEKSLKDL IKGIRSHNET PEKLDQFFKQ VLSECREEVN SPDLNSKTNA VLKLTYLEMY GFDMAWCNF HILEVMSSNK LQQKRVGYLA ASQSFYKDSD ILMLATNLLK KDLKYDGNND VVKVGIALSG LSTIITPSLA R DIADDLFT ...String:
MTSLYAPGAE DIRQRLRPFG FFFEKSLKDL IKGIRSHNET PEKLDQFFKQ VLSECREEVN SPDLNSKTNA VLKLTYLEMY GFDMAWCNF HILEVMSSNK LQQKRVGYLA ASQSFYKDSD ILMLATNLLK KDLKYDGNND VVKVGIALSG LSTIITPSLA R DIADDLFT MLNSTRPYIR KKAITALFKV FLQYPEALRD NFDKFVSKLD DDDISVVSAA VSVICELSKK NPQPFIQLSP LL YEILVTI DNNWIIIRLL KLFTNLSQVE PKLRAKLLPK ILELMESTVA TSVIYESVNC IVKGNMLEED DFETAMACLE RLH TFCDSQ DPNLRYISCI LFYKIGKINT DFISRFDQLI IRLLSDVDVS IRSKAIELVE GIVDEDNLKA IVQTLMKQFV DEDV VILQT GSIVYEKSKR IPIIIPENYK IKMVNVIISI CSADNYSSVN DFEWYNAVIM DLAMLCQDIS DKSLGSKIGE QFRNL MIKV PSMREVTIAN IIKLISNDNI NKQLPTVLRE CIWCLGEFST LVENGNDLIK IMTENISYYS HSVQEVLILA LVKVFS NWC NNFQEDKRFE IKMVLKELIE FFENLSYSST FEVQERSVEV LEFLRLSLEA LEEDTEGLPM LLSEVLPSFF NAYELAP IA RGTQLKLAVD ENLDLETPFL TKEAADELLD EQKSDAISDL MSDISMDEQV ELKFVDDSDT SYEEKEKLDD FENPFEIE R EKERMSNPYY LGEEDEERTK NSKDLLDLNE EESSDKKPET IRLNRTDNSL NSLSLSTTEI SRKKKKGKKK NRVQVLSDE PVIEAAPKRK DAFQKPHDNH STQNPLKKDK INLRMHSQLE NFDFSNFGQS SNAGRGSQEE GNLRKEDELE LSRLEANLIV KDEKDNLSD TEEVIVIKKK KKGKKSKSKN KLKTKAKNSP EPNEFLRDQS TDIRTLQVDG SDYKDDDDKD YKDDDDKDYK D DDDK

UniProtKB: AP-3 complex subunit delta

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Macromolecule #2: Y55_G0035830.mRNA.1.CDS.1

MacromoleculeName: Y55_G0035830.mRNA.1.CDS.1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 91.712016 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVDSIHRIAS ALDTAKVITR EAAAVATSKL GESSYTYYSQ NINPQQLVTL LNSRNSREVR DAMKRIISIM ASDDDSIDVQ LYFADVVKN ITTNDTKVKR LIHLYLLRFA ENDPNLTLLS INSLQKSLSD SNSELRCFAL SALSDMKMSS LAPIILHTVK K LVTDPSAM ...String:
MVDSIHRIAS ALDTAKVITR EAAAVATSKL GESSYTYYSQ NINPQQLVTL LNSRNSREVR DAMKRIISIM ASDDDSIDVQ LYFADVVKN ITTNDTKVKR LIHLYLLRFA ENDPNLTLLS INSLQKSLSD SNSELRCFAL SALSDMKMSS LAPIILHTVK K LVTDPSAM VRGEVALAII KLYRAGKNDY HEELLDILKE LMADTDPKVI SCAVLAYKEC YADHLELLHG HFRRYCRIIK QL DSWSQSY LIELLIKYCK QYLPKPTVVD KSSEGSPRSC PLPDKYNEIE YPSYEVVNDP DLDLFLQSLN CLIYSSNPTV ILS CCNALY QLASPLQMKN TKFIEALVRT VTMTENQGNK EMLLQAIHFL SILDQTLFLP YTKKFYVFPK DPIVASIWKI QILS TLINE SNVKEIFKEL KYYVASAHFP ENVVIMAVKS LSRCGQLSTS WESHVMKWLI DHMESHNLSA SVLDAYVNVI RMLVQ KNPT KHLRIIFKLA DLLTVQTSLA DNARAGIVWL FGEIASIEFK ICPDVLRRLI QNFSNEGPET RCQILVLSAK LLSYDI DNF KQAQVTGSEE NNQNPPYYDF SGSRISQMYN AVLYLAKYDD EFDIRDRARM ISSLFDSGKY EIVSLLLQAP KPTARSD DF IVSARLETHT PEIKEFFRML PWNTEITEVG ETGNDIREGA ELKDYNKYKK SFSSQSFITN NSARSFTSSS NAKLTGIN D GDSNSISGKG NVNTFTSQNG KKYRLQSLDE FFSDIPERKS KPRKIIKVVE ESSDEDEDES EESSDDDEYS DSSLGTSSS GTSSSHLEL

UniProtKB: Y55_G0035830.mRNA.1.CDS.1

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Macromolecule #3: AP complex subunit sigma

MacromoleculeName: AP complex subunit sigma / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 21.951646 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MIHAVLIFNK KCQPRLVKFY TPVDLPKQKL LLEQVYELIS QRNSDFQSSF LVTPPSLLLS NENNNDEVNN EDIQIIYKNY ATLYFTFIV DDQESELAIL DLIQTFVESL DRCFTEVNEL DLIFNWQTLE SVLEEIVQGG MVIETNVNRI VASVDELNKA A ESTDSKIG ...String:
MIHAVLIFNK KCQPRLVKFY TPVDLPKQKL LLEQVYELIS QRNSDFQSSF LVTPPSLLLS NENNNDEVNN EDIQIIYKNY ATLYFTFIV DDQESELAIL DLIQTFVESL DRCFTEVNEL DLIFNWQTLE SVLEEIVQGG MVIETNVNRI VASVDELNKA A ESTDSKIG RLTSTGFGSA LQAFAQGGFA QWATGQ

UniProtKB: AP complex subunit sigma

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Macromolecule #4: AP-3 complex subunit mu

MacromoleculeName: AP-3 complex subunit mu / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 54.899062 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MYLSFYITDT KNKLIFQYLL GATAPSFKHL WTRVQSTCPQ LLEDSSSDDY LDHSMVGRDL EVYKYFSVIN KLNYWCLAST SKSKGPLDC FTFLETIDRI LLEYFDKDKL SIKKIVNNYD RISLIFNCCV EAGEPNVSDM LYVNKIKEAV PERSDLSKFI S STAHNLQQ ...String:
MYLSFYITDT KNKLIFQYLL GATAPSFKHL WTRVQSTCPQ LLEDSSSDDY LDHSMVGRDL EVYKYFSVIN KLNYWCLAST SKSKGPLDC FTFLETIDRI LLEYFDKDKL SIKKIVNNYD RISLIFNCCV EAGEPNVSDM LYVNKIKEAV PERSDLSKFI S STAHNLQQ AVQLPQQRQQ QLQQNQISRG SNSLIENEEI VPWRTSRASK HENNELYVDL LETFHVVFEK KKSHLRLLTG SI HGIVDVR SYLNDNPLVA VKLNTMGNDI GIPSLHDCVE INDGVFSPSN ITFIPPDGKF RLLEYSVDLS SQVKQSGVRM NSI GLMSLH FQNGLGKDSD EFELSLNIEN FKKVSQVDDL KIDLQFNVEN ADPNEIAYKI KILRNTHGRF ENSIIMGQGQ WIFD KSTAT GTVPVLRGCI EYENTGPNFT KKVDLQTVSL EYSYIGQSAS GIYVEAIDIV SGLTIGKNTK LYKGAKYKTQ TGNFQ VRL

UniProtKB: AP-3 complex subunit mu

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.4 / Details: 20 mM Hepes pH 7.4 150 mM NaCl 1.5 mM MgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III
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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 81.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 958892
Startup modelType of model: OTHER / Details: Ab initio Model generated in RVIPER (SPHIRE)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPHIRE (ver. 1.3) / Software - details: MERIDIEN / Number images used: 23039
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPHIRE (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: SPHIRE (ver. 1.3) / Software - details: MERIDIEN
Final 3D classificationNumber classes: 20 / Avg.num./class: 20000 / Details: cryoDRGN v2.1.0

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7p3x:
Homology model of the full-length AP-3 complex in a compact open conformation

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