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- PDB-5yfp: Cryo-EM Structure of the Exocyst Complex -

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Entry
Database: PDB / ID: 5yfp
TitleCryo-EM Structure of the Exocyst Complex
Components(Exocyst complex component ...) x 8
KeywordsEXOCYTOSIS / exocyst / coiled-coil
Function / homology
Function and homology information


vesicle tethering involved in exocytosis / exocyst assembly / exocyst localization / negative regulation of SNARE complex assembly / endoplasmic reticulum inheritance / exocyst / prospore membrane / incipient cellular bud site / cellular bud tip / Golgi inheritance ...vesicle tethering involved in exocytosis / exocyst assembly / exocyst localization / negative regulation of SNARE complex assembly / endoplasmic reticulum inheritance / exocyst / prospore membrane / incipient cellular bud site / cellular bud tip / Golgi inheritance / cellular bud neck / Golgi to plasma membrane transport / mating projection tip / vesicle docking involved in exocytosis / spliceosomal complex assembly / exocytosis / transport vesicle / Rho protein signal transduction / phosphatidylinositol-4,5-bisphosphate binding / SNARE binding / cell periphery / intracellular protein transport / small GTPase binding / intracellular protein localization / protein transport / plasma membrane / cytoplasm
Similarity search - Function
Exocyst complex component EXOC6/Sec15 / Exocyst complex component Sec10-like / Exocyst complex component EXOC6/Sec15, C-terminal, domain 1 / Exocyst complex subunit Sec15, C-terminal / : / : / : / Exocyst complex subunit Sec15 C-terminal / Exocyst complex component Sec10-like, alpha-helical bundle / Exocyst complex component EXOC6/Sec15, N-terminal ...Exocyst complex component EXOC6/Sec15 / Exocyst complex component Sec10-like / Exocyst complex component EXOC6/Sec15, C-terminal, domain 1 / Exocyst complex subunit Sec15, C-terminal / : / : / : / Exocyst complex subunit Sec15 C-terminal / Exocyst complex component Sec10-like, alpha-helical bundle / Exocyst complex component EXOC6/Sec15, N-terminal / Exocyst complex component Sec10, N-terminal / PH domain found in exocyst complex component Exo84 / Exocyst complex component Sec8, N-terminal / Exocyst complex component EXOC3/Sec6 / Exocyst complex component EXOC2/Sec5 / Exocyst complex component EXOC2/Sec5, N-terminal domain / Exocyst complex component Sec8/EXOC4 / Exocyst complex component EXOC3/Sec6, C-terminal domain / : / Exocyst complex component Sec8 N-terminal / Exocyst complex component Sec6 / Exocyst complex component Sec5 / Exocyst complex component Sec8 C-terminal / Exocyst component Exo84, C-terminal / Exocyst complex component Exo84 / Exocyst component Exo84, C-terminal, subdomain 2 / Exocyst component Exo84, C-terminal, subdomain 1 / Exocyst component 84 C-terminal / Exocyst complex component Sec3, C-terminal / Exocyst complex component Sec3, PIP2-binding N-terminal domain / : / Exocyst complex component Sec3, coiled-coil / Exocyst complex component SEC3 N-terminal PIP2 binding PH / Exocyst complex component Sec3, C-terminal / Exocyst complex component SEC3 N-terminal PIP2 binding PH / Exocyst complex component Exo70 / EXOC6/PINT-1/Sec15/Tip20, C-terminal, domain 2 / Exocyst complex subunit Exo70, C-terminal / Exo70 exocyst complex subunit C-terminal / Vps51/EXO84/COG1 N-terminal / Exocyst complex component Exo70 N-terminal / Cullin repeat-like-containing domain superfamily / PH-like domain superfamily
Similarity search - Domain/homology
Exocyst complex component EXO70 / Exocyst complex component SEC15 / Exocyst complex component SEC6 / Exocyst complex component SEC8 / Exocyst complex component SEC3 / Exocyst complex component EXO84 / Exocyst complex component SEC5 / Exocyst complex component SEC10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Saccharomyces cerevisia S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsMei, K. / Li, Y. / Wang, S. / Shao, G. / Wang, J. / Ding, Y. / Luo, G. / Yue, P. / Liu, J.J. / Wang, X. ...Mei, K. / Li, Y. / Wang, S. / Shao, G. / Wang, J. / Ding, Y. / Luo, G. / Yue, P. / Liu, J.J. / Wang, X. / Dong, M.Q. / Guo, W. / Wang, H.W.
Funding support China, United States, 4items
OrganizationGrant numberCountry
National Science Foundation of China31530018 China
Beijing Municipal Science & Technology CommissionZ161100000116034 China
National Key Research and Development Program of MOST2016YFA0501100 China
National Institute of HealthR01GM111128 United States
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Cryo-EM structure of the exocyst complex.
Authors: Kunrong Mei / Yan Li / Shaoxiao Wang / Guangcan Shao / Jia Wang / Yuehe Ding / Guangzuo Luo / Peng Yue / Jun-Jie Liu / Xinquan Wang / Meng-Qiu Dong / Hong-Wei Wang / Wei Guo /
Abstract: The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many ...The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
History
DepositionSep 21, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 31, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type
Revision 1.4Nov 13, 2024Group: Data collection / Source and taxonomy / Structure summary
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_molwt / em_entity_assembly_naturalsource / entity_src_nat / pdbx_entry_details
Item: _em_admin.last_update / _entity_src_nat.pdbx_organism_scientific / _entity_src_nat.strain

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-6827
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Exocyst complex component SEC3
B: Exocyst complex component SEC5
C: Exocyst complex component SEC6
D: Exocyst complex component SEC8
E: Exocyst complex component SEC10
F: Exocyst complex component SEC15
G: Exocyst complex component EXO70
H: Exocyst complex component EXO84


Theoretical massNumber of molelcules
Total (without water)845,6918
Polymers845,6918
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The elution position of the exocyst complex is around 1MD, and SDS-PAGE of the gel filtration fractions shows that all subunits are present in a similar ratio., microscopy, ...Evidence: gel filtration, The elution position of the exocyst complex is around 1MD, and SDS-PAGE of the gel filtration fractions shows that all subunits are present in a similar ratio., microscopy, Both negative staining and Cryo microscope analysis show that the exocyst complex is well folded and properly assembled.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Exocyst complex component ... , 8 types, 8 molecules ABCDEFGH

#1: Protein Exocyst complex component SEC3 / Protein PSL1


Mass: 154889.547 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast) / Strain: S288c / References: UniProt: P33332
#2: Protein Exocyst complex component SEC5


Mass: 112236.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: P89102
#3: Protein Exocyst complex component SEC6


Mass: 93539.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: P32844
#4: Protein Exocyst complex component SEC8


Mass: 122367.109 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: P32855
#5: Protein Exocyst complex component SEC10


Mass: 100459.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: Q06245
#6: Protein Exocyst complex component SEC15


Mass: 105166.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: P22224
#7: Protein Exocyst complex component EXO70 / Exocyst complex protein of 70 kDa


Mass: 71382.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisia S288c (yeast) / Strain: S288c / References: UniProt: P19658
#8: Protein Exocyst complex component EXO84 / Exocyst complex protein of 84 kDa / U1 SNP1-associating protein 3


Mass: 85649.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast) / Strain: S288c / References: UniProt: P38261

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Details

Has protein modificationN

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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

ComponentName: Cryo-EM map of the Yeast Exocyst complex overall structure at 5.5A resolution
Type: COMPLEX / Details: overall map of Yeast Exocyst complex / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.844 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisia S288c (yeast) / Strain: S288c / Cellular location: cytoplasm
Buffer solutionpH: 7.4
Details: Solutions were freshly prepared from stock and filtered with 0.22um membrane to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
210 mMHEPESC8H18N204S1
32 mMDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: Purchased Quantifoil R1.2/1.3 gold grid was directly glow discharged 60 seconds before use. Before glow discharge, the sample chamber was vacuumed by an air bump for 2 minutes.
Grid material: GOLD / 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: 301 K
Details: Protein sample was applied onto a Quantifoil R1.2/1.3 golden grid and hold for 60 seconds, then blot for 2.5 seconds before plunging.

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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 magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 1800 nm / Calibrated defocus max: 3200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 123 K / Temperature (min): 113 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 11 / Num. of real images: 6466
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 0-31

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryFitting-IDDetails
1RELION1.4particle selection
2UCSFImage4image acquisition
4RELION1.4CTF correction
7Situs2.8model fitting1
8Chimera1.10.1model fitting1
10PHENIX1.11.1model refinement1
11Situs2.8model fitting2
12PHENIX1.11.1model refinement2
13Chimera1.10.1model fitting3
14PHENIX1.11.1model refinement3
15Chimera1.10.1model fitting4
16Coot0.8.2model refinement4EL
17PHENIX1.11.1model refinement4
18Chimera1.10.1model fitting5
19PHENIX1.11.1model refinement5
20Chimera1.10.1model fitting6
21Coot0.8.2model refinement6
22PHENIX1.11.1model refinement6
23Coot0.8.2model fitting7
24PHENIX1.11.1model refinement7
25RELION1.4initial Euler assignment
26RELION1.4final Euler assignment
27RELION1.4classification
28RELION1.43D reconstruction
Image processingDetails: The selected image stacks were motion corrected by Motioncor2 software and distortion magnification corrected by script.
CTF correctionDetails: The defocus value of each image was determined by CTFFIND3.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 904481 / Details: Semi-auto-picked by RELION1.4.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 343342 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALExo70 (a.a.67-623) was first fit into the map with Situs as a rigid body. Then it was split as Exo70 (a.a.67-344) and Exo70 (a.a.345-623), and fit into the same position with Chimera.
2RIGID BODY FITREALWell fit by one step Situs fitting.
3RIGID BODY FITREALThe fitting position of this chain was determined by cross-link mass spectrum.
4RIGID BODY FITREALThe yeast Sec10 (a.a.234-867) model was first obtained with Swiss-Model prediction based on this zebrafish originated initial model, and then fit into the map. Local adjustment was made with Coot. Final refinement was proceeded with Phenix real-space refinement.
5RIGID BODY FITREALThe yeast Exo84 (a.a.346-470) model was first obtained with PHYRE2 prediction based on this rat originated initial model, and then fit into the map. Final refinement was proceeded with Phenix real-space refinement.
6RIGID BODY FITREALThe yeast Sec15 (a.a.482-896) model was first obtained with PHYRE2 prediction based on this Drosophila originated initial model, and then fit into the map. Local adjustment was made with Coot. Final refinement was proceeded with Phenix real-space refinement.
7BACKBONE TRACEREALAll remaining models were manually built as backbone tracing Poly-Alanine, yet the present residues are shown as original. The backbone trace was guided by the EM map connectivity, secondary structure prediction and cross-link mass spectrum. The model was validated with different sets of cross-link mass spectrum data to confirmed the correctness.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-IDPdb chain residue rangeSource nameType
12B1E

2b1e

A12B1E167-623PDBexperimental model
22D2S

2d2s

A22D2S2525-753PDBexperimental model
32FJI

2fji

A32FJI3411-805PDBexperimental model
45H11

5h11

A45H114195-708PDBexperimental model
51ZC4

1zc4

B51ZC45171-283PDBexperimental model
62A2F

2a2f

X62A2F6383-699PDBexperimental model
77
RefinementHighest resolution: 4.4 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00633268
ELECTRON MICROSCOPYf_angle_d1.33145981
ELECTRON MICROSCOPYf_dihedral_angle_d7.02519874
ELECTRON MICROSCOPYf_chiral_restr0.0566126
ELECTRON MICROSCOPYf_plane_restr0.0066412

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