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- EMDB-28743: Cryo-EM structure of the S. cerevisiae Arf-like protein Arl1 boun... -

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Basic information

Entry
Database: EMDB / ID: EMD-28743
TitleCryo-EM structure of the S. cerevisiae Arf-like protein Arl1 bound to the Arf guanine nucleotide exchange factor Gea2
Map data
Sample
  • Complex: Gea2 homodimer in complex with two Arl1 monomers
    • Protein or peptide: ARF guanine-nucleotide exchange factor 2
    • Protein or peptide: ADP-ribosylation factor-like protein 1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
Keywordssmall GTPase / guanine nucleotide exchange factor / membrane trafficking / lipid flippase / trans-Golgi network / protein transport / LIPID TRANSPORT
Function / homology
Function and homology information


Retrograde transport at the Trans-Golgi-Network / secretory granule organization / VxPx cargo-targeting to cilium / trans-Golgi network membrane organization / Golgi cis cisterna / trans-Golgi Network Vesicle Budding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / regulation of ARF protein signal transduction / cytoplasm to vacuole targeting by the Cvt pathway ...Retrograde transport at the Trans-Golgi-Network / secretory granule organization / VxPx cargo-targeting to cilium / trans-Golgi network membrane organization / Golgi cis cisterna / trans-Golgi Network Vesicle Budding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / regulation of ARF protein signal transduction / cytoplasm to vacuole targeting by the Cvt pathway / protein localization to Golgi apparatus / Golgi to plasma membrane protein transport / intra-Golgi vesicle-mediated transport / protein localization to phagophore assembly site / protein targeting to vacuole / protein-containing complex localization / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / response to endoplasmic reticulum stress / guanyl-nucleotide exchange factor activity / macroautophagy / intracellular protein transport / trans-Golgi network / endocytosis / actin cytoskeleton organization / endosome membrane / Golgi membrane / GTPase activity / GTP binding / Golgi apparatus / cytosol
Similarity search - Function
Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases ...Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / 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 / ARF-like small GTPases; ARF, ADP-ribosylation factor / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADP-ribosylation factor-like protein 1 / ARF guanine-nucleotide exchange factor 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDuan HD / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin.
Authors: H Diessel Duan / Bhawik K Jain / Hua Li / Todd R Graham / Huilin Li /
Abstract: Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates ...Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.
History
DepositionNov 1, 2022-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28743.png

Downloads & links

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Map

FileDownload / File: emd_28743.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.066803776 - 2.491642
Average (Standard dev.)0.00093760254 (±0.018717043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 423.936 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Gea2 homodimer in complex with two Arl1 monomers

EntireName: Gea2 homodimer in complex with two Arl1 monomers
Components
  • Complex: Gea2 homodimer in complex with two Arl1 monomers
    • Protein or peptide: ARF guanine-nucleotide exchange factor 2
    • Protein or peptide: ADP-ribosylation factor-like protein 1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate

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Supramolecule #1: Gea2 homodimer in complex with two Arl1 monomers

SupramoleculeName: Gea2 homodimer in complex with two Arl1 monomers / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 379 KDa

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Macromolecule #1: ARF guanine-nucleotide exchange factor 2

MacromoleculeName: ARF guanine-nucleotide exchange factor 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 168.590703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNAMSDREF VTVDPVTIII KECINLSTAM RKYSKFTSQS GVAALLGGGS EIFSNQDDYL AHTFNNLNT NKHNDPFLSG FIQLRLMLNK LKNLDNIDSL TILQPFLLIV STSSISGYIT SLALDSLQKF FTLNIINESS Q NYIGAHRA ...String:
MHHHHHHSSG VDLGTENLYF QSNAMSDREF VTVDPVTIII KECINLSTAM RKYSKFTSQS GVAALLGGGS EIFSNQDDYL AHTFNNLNT NKHNDPFLSG FIQLRLMLNK LKNLDNIDSL TILQPFLLIV STSSISGYIT SLALDSLQKF FTLNIINESS Q NYIGAHRA TVNALTHCRF EGSQQLSDDS VLLKVVFLLR SIVDSPYGDL LSNSIIYDVL QTILSLACNN RRSEVLRNAA QS TMIAVTV KIFSKLKTIE PVNVNQIYIN DESYTNDVLK ADTIGTNVES KEEGSQEDPI GMKVNNEEAI SEDDGIEEEH IHS EKSTNG AEQLDIVQKT TRSNSRIQAY ADDNYGLPVV RQYLNLLLSL IAPENELKHS YSTRIFGLEL IQTALEISGD RLQL YPRLF TLISDPIFKS ILFIIQNTTK LSLLQATLQL FTTLVVILGN NLQLQIELTL TRIFSILLDD GTANNSSSEN KNKPS IIKE LLIEQISILW TRSPSFFTST FINFDCNLDR ADVSINFLKA LTKLALPESA LTTTESVPPI CLEGLVSLVD DMFDHM KDI DREEFGRQKN EMEILKKRDR KTEFIECTNA FNEKPKKGIP MLIEKGFIAS DSDKDIAEFL FNNNNRMNKK TIGLLLC HP DKVSLLNEYI RLFDFSGLRV DEAIRILLTK FRLPGESQQI ERIIEAFSSA YCENQDYDPS KISDNAEDDI STVQPDAD S VFILSYSIIM LNTDLHNPQV KEHMSFEDYS GNLKGCCNHK DFPFWYLDRI YCSIRDKEIV MPEEHHGNEK WFEDAWNNL ISSTTVITEI KKDTQSVMDK LTPLELLNFD RAIFKQVGPS IVSTLFNIYV VASDDHISTR MITSLDKCSY ISAFFDFKDL FNDILNSIA KGTTLINSSH DDELSTLAFE YGPMPLVQIK FEDTNTEIPV STDAVRFGRS FKGQLNTVVF FRIIRRNKDP K IFSKELWL NIVNIILTLY EDLILSPDIF PDLQKRLKLS NLPKPSPEIS INKSKESKGL LSTFASYLKG DEEPTEEEIK SS KKAMECI KSSNIAASVF GNESNITADL IKTLLDSAKT EKNADNSRYF EAELLFIIEL TIALFLFCKE EKELGKFILQ KVF QLSHTK GLTKRTVRRM LTYKILLISL CADQTEYLSK LINDELLKKG DIFTQKFFAT NQGKEFLKRL FSLTESEFYR GFLL GNENF WKFLRKVTAM KEQSESIFEY LNESIKTDSN ILTNENFMWV LGLLDEISSM GAVGNHWEIE YKKLTESGHK IDKEN PYKK SIELSLKSIQ LTSHLLEDNN DLRKNEIFAI IQALAHQCIN PCKQISEFAV VTLEQTLINK IEIPTNEMES VEELIE GGL LPLLNSSETQ EDQKILISSI LTIISNVYLH YLKLGKTSNE TFLKILSIFN KFVEDSDIEK KLQQLILDKK SIEKGNG SS SHGSAHEQTP ESNDVEIEAT APIDDNTDDD NKPKLSDVEK D

UniProtKB: ARF guanine-nucleotide exchange factor 2

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Macromolecule #2: ADP-ribosylation factor-like protein 1

MacromoleculeName: ADP-ribosylation factor-like protein 1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 20.460168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ELRILILGLD GAGKTTILYR LQIGEVVTTK PTIGFNVETL SYKNLKLNVW DLGGLTSIRP YWRCYYADTA AVIFVVDSTD KDRMSTASK ELHLMLQEEE LQDAALLVFA NKQDQPGALS ASEVSKELNL VELKDRSWSI VASSAIKGEG ITEGLDWLID V IKEEQLAG ENLYFQSAGH HHHHH

UniProtKB: ADP-ribosylation factor-like protein 1

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.3 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 69.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / Details: PDB 5EE5 was also used as a startup model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 712548

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