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- PDB-8eki: CryoEM structure of the Dsl1 complex bound to SNAREs Sec20 and Use1 -

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Basic information

Entry
Database: PDB / ID: 8eki
TitleCryoEM structure of the Dsl1 complex bound to SNAREs Sec20 and Use1
Components
  • Protein transport protein DSL1
  • Protein transport protein SEC20
  • Protein transport protein SEC39
  • Protein transport protein TIP20
  • Protein transport protein USE1
KeywordsTRANSPORT PROTEIN / Tether / SNARE / Complex
Function / homology
Function and homology information


Golgi to ER transport vesicle membrane / vesicle fusion with endoplasmic reticulum / ER-dependent peroxisome organization / Dsl1/NZR complex / RZZ complex / COPI-dependent Golgi-to-ER retrograde traffic / regulation of ER to Golgi vesicle-mediated transport / SNAP receptor activity / SNARE complex / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum ...Golgi to ER transport vesicle membrane / vesicle fusion with endoplasmic reticulum / ER-dependent peroxisome organization / Dsl1/NZR complex / RZZ complex / COPI-dependent Golgi-to-ER retrograde traffic / regulation of ER to Golgi vesicle-mediated transport / SNAP receptor activity / SNARE complex / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / mitotic spindle assembly checkpoint signaling / cytoplasmic side of endoplasmic reticulum membrane / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / autophagy / nuclear envelope / protein transport / endoplasmic reticulum membrane / endoplasmic reticulum / nucleus / membrane / cytosol
Similarity search - Function
Sec20 / Vesicle transport protein, Use1 / Sec20 / Membrane fusion protein Use1 / Sec39 domain / Secretory pathway protein Sec39 / RINT-1/Tip20 / Protein transport protein Tip20, domain E / Protein transport protein Tip20, domain A / Protein transport protein Tip20, domain B ...Sec20 / Vesicle transport protein, Use1 / Sec20 / Membrane fusion protein Use1 / Sec39 domain / Secretory pathway protein Sec39 / RINT-1/Tip20 / Protein transport protein Tip20, domain E / Protein transport protein Tip20, domain A / Protein transport protein Tip20, domain B / Protein transport protein Tip20, domain C / RINT-1/TIP-1 family / RINT1/TIP20 domain profile. / Retrograde transport protein Dsl1 N-terminal domain / Retrograde transport protein Dsl1, C-terminal domain / Dsl1, N-terminal domain superfamily / Retrograde transport protein Dsl1 N terminal / Retrograde transport protein Dsl1 C terminal / EXOC6/PINT-1/Sec15/Tip20, C-terminal, domain 2 / Zw10/DSL1, C-terminal / Endoplasmic reticulum targeting sequence.
Similarity search - Domain/homology
Protein transport protein SEC20 / Protein transport protein TIP20 / Protein transport protein USE1 / Protein transport protein DSL1 / Protein transport protein SEC39
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsDAmico, K.A. / Jeffrey, P.D. / Hughson, F.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071574 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM007388 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structure of a membrane tethering complex incorporating multiple SNAREs.
Authors: Kevin A DAmico / Abigail E Stanton / Jaden D Shirkey / Sophie M Travis / Philip D Jeffrey / Frederick M Hughson /
Abstract: Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial ...Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here we present a cryo-electron microscopy structure of the simplest known MTC, the 255-kDa Dsl1 complex of Saccharomyces cerevisiae, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.
History
DepositionSep 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jan 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Feb 28, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein transport protein SEC20
B: Protein transport protein USE1
C: Protein transport protein TIP20
D: Protein transport protein SEC39
E: Protein transport protein DSL1


Theoretical massNumber of molelcules
Total (without water)311,6815
Polymers311,6815
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Five proteins migrate as a single pentamer when co-expressed and purified over size-exclusion chromatography., electron microscopy, Particles of the five-protein complex ...Evidence: gel filtration, Five proteins migrate as a single pentamer when co-expressed and purified over size-exclusion chromatography., electron microscopy, Particles of the five-protein complex assume a conformation that is not accessible in the absence of any given subunit.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein transport protein SEC20


Mass: 32244.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: SEC20, YDR498C, D9719.4 / Plasmid: pQLink / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P28791
#2: Protein Protein transport protein USE1 / SNARE-like tail-anchored protein 1 / Unconventional SNARE in the endoplasmic reticulum protein 1


Mass: 24254.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: USE1, SLT1, YGL098W / Plasmid: pQLink / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P53146
#3: Protein Protein transport protein TIP20


Mass: 81253.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: TIP20, TIP1, YGL145W / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P33891
#4: Protein Protein transport protein SEC39 / Dependent on SLY1-20 protein 3


Mass: 82483.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: SEC39, DSL3, YLR440C / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q12745
#5: Protein Protein transport protein DSL1 / Dependent on SLY1-20 protein 1


Mass: 91445.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: DSL1, YNL258C, N0842 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P53847

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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

Component
IDNameTypeEntity IDParent-IDSource
1Dsl1 complex bound to SNARE proteins Sec20 and Use1COMPLEXall0RECOMBINANT
2Dsl1 ComplexCOMPLEX#3-#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.31234826 MDaNO
210.25541261 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Saccharomyces cerevisiae (brewer's yeast)559292ATCC 204508/S288c
22Saccharomyces cerevisiae (brewer's yeast)559292ATCC 204508/S288c
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Escherichia coli (E. coli)511693BL21
22Escherichia coli (E. coli)511693BL21
Buffer solutionpH: 7.5
Details: Buffer was made fresh from concentrated components and sterile filtered. NP40 was not present during protein purification but was an additive during the grid preparation.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMHEPESC8H18N2O4S1
31 mMDTTC4H10O2S21
40.05 %NP40H(C2H4O)nO(C6H4)C9H191
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was consistently in the thickest regions of ice only, often close to the edges of the carbon hole
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Force=0 Wait Time=0 Blot Time=6s Drain Time=0

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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 defocus max: 2500 nm / Nominal defocus min: 1250 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5857

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Processing

SoftwareName: PHENIX / Version: 1.17_3644: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC4.1.1particle selection
2EPUimage acquisition
4cryoSPARC4.1.1CTF correction
9PHENIXmodel refinement
10cryoSPARC4.1.1initial Euler assignment
11cryoSPARC4.1.1final Euler assignment
12cryoSPARC4.1.1classification
13Coot0.8.9.13D reconstruction
CTF correctionDetails: correction was performed using the Patch CTF Estimation tool in CryoSPARC
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 469193
Details: Particles were template-picked utilizing a low-resolution density map of the complex, generated from an initial subset of the data.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49947 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00519148
ELECTRON MICROSCOPYf_angle_d0.69325820
ELECTRON MICROSCOPYf_dihedral_angle_d7.4022453
ELECTRON MICROSCOPYf_chiral_restr0.0432917
ELECTRON MICROSCOPYf_plane_restr0.0043290

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