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- EMDB-28204: CryoEM structure of the Dsl1 complex bound to SNAREs Sec20 and Use1 -

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Basic information

Entry
Database: EMDB / ID: EMD-28204
TitleCryoEM structure of the Dsl1 complex bound to SNAREs Sec20 and Use1
Map dataHalf map B
Sample
  • Complex: Dsl1 complex bound to SNARE proteins Sec20 and Use1
    • Complex: Dsl1 Complex
      • Protein or peptide: Protein transport protein TIP20
      • Protein or peptide: Protein transport protein SEC39
      • Protein or peptide: Protein transport protein DSL1
    • Protein or peptide: Protein transport protein SEC20
    • Protein or peptide: Protein transport protein USE1
KeywordsTether / SNARE / Complex / TRANSPORT PROTEIN
Function / homology
Function and homology information


Golgi to ER transport vesicle membrane / vesicle fusion with endoplasmic reticulum / ER-dependent peroxisome organization / RZZ complex / Dsl1/NZR complex / COPI-dependent Golgi-to-ER retrograde traffic / regulation of ER to Golgi vesicle-mediated transport / SNARE complex / SNAP receptor activity / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum ...Golgi to ER transport vesicle membrane / vesicle fusion with endoplasmic reticulum / ER-dependent peroxisome organization / RZZ complex / Dsl1/NZR complex / COPI-dependent Golgi-to-ER retrograde traffic / regulation of ER to Golgi vesicle-mediated transport / SNARE complex / SNAP receptor activity / 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 / protein transport / nuclear envelope / endoplasmic reticulum membrane / endoplasmic reticulum / membrane / nucleus / 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 / Zw10/DSL1, C-terminal / Retrograde transport protein Dsl1 N terminal / Retrograde transport protein Dsl1 C terminal / EXOC6/PINT-1/Sec15/Tip20, C-terminal, domain 2 / 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 (brewer's yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsDAmico KA / Jeffrey PD / Hughson FM
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28204.png

Downloads & links

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Map

FileDownload / File: emd_28204.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHalf map B
Voxel sizeX=Y=Z: 1.114 Å
Density
Contour LevelBy AUTHOR: 0.186
Minimum - Maximum-0.08617353 - 1.3723472
Average (Standard dev.)0.0077837794 (±0.021243798)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 445.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_28204_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_28204_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dsl1 complex bound to SNARE proteins Sec20 and Use1

EntireName: Dsl1 complex bound to SNARE proteins Sec20 and Use1
Components
  • Complex: Dsl1 complex bound to SNARE proteins Sec20 and Use1
    • Complex: Dsl1 Complex
      • Protein or peptide: Protein transport protein TIP20
      • Protein or peptide: Protein transport protein SEC39
      • Protein or peptide: Protein transport protein DSL1
    • Protein or peptide: Protein transport protein SEC20
    • Protein or peptide: Protein transport protein USE1

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Supramolecule #1: Dsl1 complex bound to SNARE proteins Sec20 and Use1

SupramoleculeName: Dsl1 complex bound to SNARE proteins Sec20 and Use1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c
Molecular weightTheoretical: 255.41261 KDa

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Supramolecule #2: Dsl1 Complex

SupramoleculeName: Dsl1 Complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3-#5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c

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Macromolecule #1: Protein transport protein SEC20

MacromoleculeName: Protein transport protein SEC20 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 32.244254 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MVVTFLQDLE VLQDALLNNL QKLSAISRRK ESGESKHDNK DSFAAIANEH NDEEEEIEFE DLVNIIESKV SDFESVLKCS IVEMTYKYP ELKLQWEKSP RYDQCDKLHI VKLDKQMNED IYAQLVEELD FVLQFVDWFY CYRLKVKEIL RQHHKRDLAW N DEKRDRAI ...String:
MVVTFLQDLE VLQDALLNNL QKLSAISRRK ESGESKHDNK DSFAAIANEH NDEEEEIEFE DLVNIIESKV SDFESVLKCS IVEMTYKYP ELKLQWEKSP RYDQCDKLHI VKLDKQMNED IYAQLVEELD FVLQFVDWFY CYRLKVKEIL RQHHKRDLAW N DEKRDRAI KFHAVDYDKL HQGTSSSSSL TSTSMEKAST REKLLSKTKQ LTNNLVRGNQ ILQSGILQSD LNLDELRAQT NS LTQIDDK YTQFETVFKK TADLVKVLEN ASHQEKRD

UniProtKB: Protein transport protein SEC20

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Macromolecule #2: Protein transport protein USE1

MacromoleculeName: Protein transport protein USE1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 24.254781 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MAETSNDPFL SYVLSSKQLT NLNRLRRKAV TKQLGSSDDN KVSEEFLRYQ HTYQREAFEY LQTKHDAHKI MESQYEQYQS SSKTRRYSI DLDSVDAVDT ESQTEYPNEE FIDRNEDSEA VMELRKRLLG KGQNKGLGYE TTKSVDRQIE DQDTLQQDLI Q DMSKLVGS ...String:
MAETSNDPFL SYVLSSKQLT NLNRLRRKAV TKQLGSSDDN KVSEEFLRYQ HTYQREAFEY LQTKHDAHKI MESQYEQYQS SSKTRRYSI DLDSVDAVDT ESQTEYPNEE FIDRNEDSEA VMELRKRLLG KGQNKGLGYE TTKSVDRQIE DQDTLQQDLI Q DMSKLVGS LKQGAVAFQS ALDEDKQVLG AAEIGIQVAS QGLMDVSGKL RKYD

UniProtKB: Protein transport protein USE1

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Macromolecule #3: Protein transport protein TIP20

MacromoleculeName: Protein transport protein TIP20 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 81.253062 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MNGIDDLLNI NDRIKQVQNE RNELASKLQN LKQSLASNDT EVALSEVIAQ DIIEVGASVE GLEQLRAKYG DLQILNKLEK VAVQQTQMQ AGVDKLDSFE RQLDELAEQP PDQFTLDDVK ALHSKLTSVF ATVPQINNID SQYAAYNKLK SKVTGKYNDV I IQRLATNW ...String:
MNGIDDLLNI NDRIKQVQNE RNELASKLQN LKQSLASNDT EVALSEVIAQ DIIEVGASVE GLEQLRAKYG DLQILNKLEK VAVQQTQMQ AGVDKLDSFE RQLDELAEQP PDQFTLDDVK ALHSKLTSVF ATVPQINNID SQYAAYNKLK SKVTGKYNDV I IQRLATNW SNTFDQKLLE AQWDTQKFAS TSVGLVKCLR ENSTKLYQLS LLYLPLEEET QNGDSERPLS RSNNNQEPVL WN FKSLANN FNVRFTYHFH ATSSSSKIET YFQFLNDYLA ENLYKCINIF HDDCNGLTKP VIHEQFINYV LQPIRDKVRS TLF QNDLKT LIVLISQILA TDKNLLNSFH YHGLGLVSLI SDEVWEKWIN YEVEMANRQF INITKNPEDF PKSSQNFVKL INKI YDYLE PFYDLDFDLL VRYKLMTCSL IFMNLTSSYL DYILTVDSLN ETRTKEQELY QTMAKLQHVN FVYRKIKSLS SNFIF IQLT DIVNSTESKK YNSLFQNVEN DYEKAMSTDM QNSIVHRIQK LLKETLRNYF KISTWSTLEM SVDENIGPSS VPSAEL VNS INVLRRLINK LDSMDIPLAI SLKVKNELLN VIVNYFTESI LKLNKFNQNG LNQFLHDFKS LSSILSLPSH ATNYKCM SL HELVKILKLK YDPNNQQFLN PEYIKTGNFT SLKEAYSIKY LKDTKIQDAL YRIIYGNIL

UniProtKB: Protein transport protein TIP20

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Macromolecule #4: Protein transport protein SEC39

MacromoleculeName: Protein transport protein SEC39 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 82.483797 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MLEEQLYLLA CIFASRADTR NIKKLSTRLG SQSKYLEILC VLWPELDDPK NLLFLRELEE EVQSPEGEET TDEDVIVELL ESDSSLIPL IESDTTTRSN RYHELQEFIS KKLNNKTLEN FEEWLRERIL ICNEMIPETP LLYSVLWETA KSKVLSTKFI G WVEGVLKP ...String:
MLEEQLYLLA CIFASRADTR NIKKLSTRLG SQSKYLEILC VLWPELDDPK NLLFLRELEE EVQSPEGEET TDEDVIVELL ESDSSLIPL IESDTTTRSN RYHELQEFIS KKLNNKTLEN FEEWLRERIL ICNEMIPETP LLYSVLWETA KSKVLSTKFI G WVEGVLKP LDHLNKRLHL IFKINEWEKM PDSELFKIIF DGVEDMQGYI GIADVIEDEL APTLSYGKKW ETFITEFFNK QQ FSLKSDT NYQLFIKLYY SLEKGVKDNS EASRKLQSNV VDILFHNSEN LFNLSSLTHK LDELWSILSG FPDEITIEEQ KTI TALEMK QFMEFFIKCS TKFSFKEIFA ITQEEESAQL AHFSSLCHEE FNKANEISSF LQAMYETVLD ISKDDKIFTR ISMD EKLYS ILEILLQMNE FAYIEAIIER FDYSNNTQIY ELLVKFFWHF FNNASNGLRK EPEMKKASQT LQIIQKHMSQ RAGTN LTKL EVLLEISDKL SHYSINLNKS HNGARDTAFK PSNILEYRDC PLDIISNLLE LNPRLYKDLP TTKSLLFGIY DSLSIN REG QTGKVEVDLM VLHIDYALVN LDFGTAYELG KQVFEICQEA GQHMMKALGD EHWLTFYQMG KFVDPNWVDN EIPTEII VL QMSILGRLLE VCPLEEVEIV TSQWSTLELE LSARDLVKDK YALDGQNDNK SKVGGIAREI FHNVTNF

UniProtKB: Protein transport protein SEC39

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Macromolecule #5: Protein transport protein DSL1

MacromoleculeName: Protein transport protein DSL1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 91.445133 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKHHHHHHHG AAGTSLYKKA GENLYFQGSM ESLFPNKGEI IRELLKDPLI LKNDSKRSNG SELELDSSDL LQREAILANE LNILDNLKT FLNLIKEVKT NLNILELENC YYSLQSLRKK MRNNAAYLKQ SFNFQQSIST YVDTLHLELV STLYKILTNG F WKITENSI ...String:
MKHHHHHHHG AAGTSLYKKA GENLYFQGSM ESLFPNKGEI IRELLKDPLI LKNDSKRSNG SELELDSSDL LQREAILANE LNILDNLKT FLNLIKEVKT NLNILELENC YYSLQSLRKK MRNNAAYLKQ SFNFQQSIST YVDTLHLELV STLYKILTNG F WKITENSI QFTPTVEWGK DKVHIEYDTF MDFVAQQYFP KGSLDNQAWF ILDMTSADSQ EQVRAKLNTI MKEYMNLSRI VS MIKNSIF ISGKEISYEN EKNILVFSKS SSHGQHCVST VLTSFEAVCD FMLDGLAFRD RKTLSYELGP LFNTEFTKFV KNN ASIILE SLDSPLKNLV SVINNKLTRL VAKSEVTNWT HSGKEIQDLL MNKQLYYNLL LDKVLESHIS EIRSIFEDPK KSWQ NLEVV ELTTSNTNTM SEKIGKNDSD VQNEKELHNA VSKDDDWNWE VEDDDADAWG DEIDVNIDDE EEKTNQEKEK EPEEE ENAW DEAWAIDENI DDASLENGKE HLKAHDVGSL DKDHIEVTQL PKLFLAISQN FKSSFADSHV DEQYFAYKYN LLQTSY MAM CTANFSHNWC QLYVDMRYLI ERDEKLYRIK ELTRNLLETK LNMKYRIVCQ LIRHQLTEFR ENERNPSWDA TIEKLLP YI LKEIVRPLQK IRGEEGSRYL LSFLNFLYND CVTKEILKWQ IISEVNSENL GELVSLLVNN TDIQLLAKEP SYKKMREK F ATMGKFLPLH LKEIMEMFYN GDFYLFATDE LIQWIELLFA DTPLRRNAID DIYEIRGTAL DD

UniProtKB: Protein transport protein DSL1

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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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium Chloride
20.0 mMC8H18N2O4SHEPES
1.0 mMC4H10O2S2DTT
0.05 %H(C2H4O)nO(C6H4)C9H19NP40

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.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Force=0 Wait Time=0 Blot Time=6s Drain Time=0.
DetailsSample was consistently in the thickest regions of ice only, often close to the edges of the carbon hole

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 5857 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.25 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 469193
Details: Particles were template-picked utilizing a low-resolution density map of the complex, generated from an initial subset of the data.
Startup modelType of model: NONE
Details: EM density was built without a reference model, and was generated only from refining classes of selected particles
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Coot (ver. 0.8.9.1) / Number images used: 49947
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 13070 / Software - Name: cryoSPARC (ver. 4.1.1)
FSC plot (resolution estimation)

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