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- EMDB-29447: Sec39:Use1:Sec20:Tip20 Local Refinement of the Dsl1:Qb:Qc Complex #2 -

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Basic information

Entry
Database: EMDB / ID: EMD-29447
TitleSec39:Use1:Sec20:Tip20 Local Refinement of the Dsl1:Qb:Qc Complex #2
Map dataSharpened Map, Local Refinement #1, of the Dsl1:Qb:Qc complex
Sample
  • Complex: Dsl1 complex bound to SNARE proteins Sec20 and Use1
    • Other: Sec39
    • Other: Use1
    • Other: Sec20
    • Other: Tip20
KeywordsTether / SNARE / Complex / TRANSPORT PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's 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: TO BE PUBLISHED
Title: CryoEM structure of the Dsl1 complex bound to SNAREs Sec20 and Use1
Authors: DAmico KA / Stanton AE / Shirkey JD / Jeffrey PD / Hughson FM
History
DepositionJan 12, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29447.png

Downloads & links

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Map

FileDownload / File: emd_29447.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map, Local Refinement #1, of the Dsl1:Qb:Qc complex
Voxel sizeX=Y=Z: 1.114 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.30600005 - 0.9056255
Average (Standard dev.)0.00010462194 (±0.016017243)
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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Mask #1

Fileemd_29447_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened Map, Local Refinement #1, of the Dsl1:Qb:Qc complex

Fileemd_29447_additional_1.map
AnnotationUnsharpened Map, Local Refinement #1, of the Dsl1:Qb:Qc complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-Map A, Local Refinement #1, of the Dsl1:Qb:Qc complex

Fileemd_29447_half_map_1.map
AnnotationHalf-Map A, Local Refinement #1, of the Dsl1:Qb:Qc complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-Map B, Local Refinement #1, of the Dsl1:Qb:Qc complex

Fileemd_29447_half_map_2.map
AnnotationHalf-Map B, Local Refinement #1, of the Dsl1:Qb:Qc complex
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
    • Other: Sec39
    • Other: Use1
    • Other: Sec20
    • Other: Tip20

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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: 312.34826 KDa

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Macromolecule #1: Sec39

MacromoleculeName: Sec39 / type: other / ID: 1 / Classification: other
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c
SequenceString: MLEEQLYLLA CIFASRADTR NIKKLSTRLG SQSKYLEILC VLWPELDDPK NLLFLRELEE EVQSPEGEET TDEDVIVELL ESDSSLIPLI ESDTTTRSNR YHELQEFISK KLNNKTLENF EEWLRERILI CNEMIPETPL LYSVLWETAK SKVLSTKFIG WVEGVLKPLD ...String:
MLEEQLYLLA CIFASRADTR NIKKLSTRLG SQSKYLEILC VLWPELDDPK NLLFLRELEE EVQSPEGEET TDEDVIVELL ESDSSLIPLI ESDTTTRSNR YHELQEFISK KLNNKTLENF EEWLRERILI CNEMIPETPL LYSVLWETAK SKVLSTKFIG WVEGVLKPLD HLNKRLHLIF KINEWEKMPD SELFKIIFDG VEDMQGYIGI ADVIEDELAP TLSYGKKWET FITEFFNKQQ FSLKSDTNYQ LFIKLYYSLE KGVKDNSEAS RKLQSNVVDI LFHNSENLFN LSSLTHKLDE LWSILSGFPD EITIEEQKTI TALEMKQFME FFIKCSTKFS FKEIFAITQE EESAQLAHFS SLCHEEFNKA NEISSFLQAM YETVLDISKD DKIFTRISMD EKLYSILEIL LQMNEFAYIE AIIERFDYSN NTQIYELLVK FFWHFFNNAS NGLRKEPEMK KASQTLQIIQ KHMSQRAGTN LTKLEVLLEI SDKLSHYSIN LNKSHNGARD TAFKPSNILE YRDCPLDIIS NLLELNPRLY KDLPTTKSLL FGIYDSLSIN REGQTGKVEV DLMVLHIDYA LVNLDFGTAY ELGKQVFEIC QEAGQHMMKA LGDEHWLTFY QMGKFVDPNW VDNEIPTEII VLQMSILGRL LEVCPLEEVE IVTSQWSTLE LELSARDLVK DKYALDGQND NKSKVGGIAR EIFHNVTNF
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: Use1

MacromoleculeName: Use1 / type: other / ID: 2 / Classification: other
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c
SequenceString: MAETSNDPFL SYVLSSKQLT NLNRLRRKAV TKQLGSSDDN KVSEEFLRYQ HTYQREAFEY LQTKHDAHKI MESQYEQYQS SSKTRRYSID LDSVDAVDTE SQTEYPNEEF IDRNEDSEAV MELRKRLLGK GQNKGLGYET TKSVDRQIED QDTLQQDLIQ DMSKLVGSLK ...String:
MAETSNDPFL SYVLSSKQLT NLNRLRRKAV TKQLGSSDDN KVSEEFLRYQ HTYQREAFEY LQTKHDAHKI MESQYEQYQS SSKTRRYSID LDSVDAVDTE SQTEYPNEEF IDRNEDSEAV MELRKRLLGK GQNKGLGYET TKSVDRQIED QDTLQQDLIQ DMSKLVGSLK QGAVAFQSAL DEDKQVLGAA EIGIQVASQG LMDVSGKLRK YD

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Macromolecule #3: Sec20

MacromoleculeName: Sec20 / type: other / ID: 3 / Classification: other
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c
SequenceString: MVVTFLQDLE VLQDALLNNL QKLSAISRRK ESGESKHDNK DSFAAIANEH NDEEEEIEFE DLVNIIESKV SDFESVLKCS IVEMTYKYPE LKLQWEKSPR YDQCDKLHIV KLDKQMNEDI YAQLVEELDF VLQFVDWFYC YRLKVKEILR QHHKRDLAWN DEKRDRAIKF ...String:
MVVTFLQDLE VLQDALLNNL QKLSAISRRK ESGESKHDNK DSFAAIANEH NDEEEEIEFE DLVNIIESKV SDFESVLKCS IVEMTYKYPE LKLQWEKSPR YDQCDKLHIV KLDKQMNEDI YAQLVEELDF VLQFVDWFYC YRLKVKEILR QHHKRDLAWN DEKRDRAIKF HAVDYDKLHQ GTSSSSSLTS TSMEKASTRE KLLSKTKQLT NNLVRGNQIL QSGILQSDLN LDELRAQTNS LTQIDDKYTQ FETVFKKTAD LVKVLENASH QEKRD

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Macromolecule #4: Tip20

MacromoleculeName: Tip20 / type: other / ID: 4 / Classification: other
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508/S288c
SequenceString: MNGIDDLLNI NDRIKQVQNE RNELASKLQN LKQSLASNDT EVALSEVIAQ DIIEVGASVE GLEQLRAKYG DLQILNKLEK VAVQQTQMQA GVDKLDSFER QLDELAEQPP DQFTLDDVKA LHSKLTSVFA TVPQINNIDS QYAAYNKLKS KVTGKYNDVI IQRLATNWSN ...String:
MNGIDDLLNI NDRIKQVQNE RNELASKLQN LKQSLASNDT EVALSEVIAQ DIIEVGASVE GLEQLRAKYG DLQILNKLEK VAVQQTQMQA GVDKLDSFER QLDELAEQPP DQFTLDDVKA LHSKLTSVFA TVPQINNIDS QYAAYNKLKS KVTGKYNDVI IQRLATNWSN TFDQKLLEAQ WDTQKFASTS VGLVKCLREN STKLYQLSLL YLPLEEETQN GDSERPLSRS NNNQEPVLWN FKSLANNFNV RFTYHFHATS SSSKIETYFQ FLNDYLAENL YKCINIFHDD CNGLTKPVIH EQFINYVLQP IRDKVRSTLF QNDLKTLIVL ISQILATDKN LLNSFHYHGL GLVSLISDEV WEKWINYEVE MANRQFINIT KNPEDFPKSS QNFVKLINKI YDYLEPFYDL DFDLLVRYKL MTCSLIFMNL TSSYLDYILT VDSLNETRTK EQELYQTMAK LQHVNFVYRK IKSLSSNFIF IQLTDIVNST ESKKYNSLFQ NVENDYEKAM STDMQNSIVH RIQKLLKETL RNYFKISTWS TLEMSVDENI GPSSVPSAEL VNSINVLRRL INKLDSMDIP LAISLKVKNE LLNVIVNYFT ESILKLNKFN QNGLNQFLHD FKSLSSILSL PSHATNYKCM SLHELVKILK LKYDPNNQQF LNPEYIKTGN FTSLKEAYSI KYLKDTKIQD ALYRIIYGNI L

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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
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 reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 49946
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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