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- PDB-6vkl: Negative stain reconstruction of the yeast exocyst octameric complex. -

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Entry
Database: PDB / ID: 6vkl
TitleNegative stain reconstruction of the yeast exocyst octameric complex.
Components(Exocyst complex component ...Exocyst) x 8
KeywordsEXOCYTOSIS / EXOCYST / COILED-COIL
Function / homology
Function and homology information


exocyst assembly / negative regulation of SNARE complex assembly / vesicle tethering involved in exocytosis / exocyst localization / endoplasmic reticulum inheritance / Golgi inheritance / exocyst / prospore membrane / GTP-Rho binding / incipient cellular bud site ...exocyst assembly / negative regulation of SNARE complex assembly / vesicle tethering involved in exocytosis / exocyst localization / endoplasmic reticulum inheritance / Golgi inheritance / exocyst / prospore membrane / GTP-Rho binding / incipient cellular bud site / cellular bud tip / Golgi to plasma membrane transport / cellular bud neck / vesicle docking involved in exocytosis / mating projection tip / protein targeting to membrane / spliceosomal complex assembly / exocytosis / Rho protein signal transduction / Rab GTPase binding / SNARE binding / transport vesicle / vesicle-mediated transport / phosphatidylinositol-4,5-bisphosphate binding / cell periphery / protein localization / protein transport / go:0005623: / membrane / plasma membrane / cytoplasm
Exocyst complex component Exo84 / Cullin repeat-like-containing domain superfamily / Exocyst complex component Exo70 / Exocyst complex component EXOC2/Sec5, N-terminal domain / Sec8 exocyst complex component specific domain / Exocyst complex component Sec8/EXOC4 / Exocyst complex component EXOC6/Sec15, C-terminal, domain 2 / Exocyst complex component EXOC6/Sec15, C-terminal, domain 1 / Exocyst complex component EXOC6/Sec15 / Exocyst complex component Sec10-like ...Exocyst complex component Exo84 / Cullin repeat-like-containing domain superfamily / Exocyst complex component Exo70 / Exocyst complex component EXOC2/Sec5, N-terminal domain / Sec8 exocyst complex component specific domain / Exocyst complex component Sec8/EXOC4 / Exocyst complex component EXOC6/Sec15, C-terminal, domain 2 / Exocyst complex component EXOC6/Sec15, C-terminal, domain 1 / Exocyst complex component EXOC6/Sec15 / Exocyst complex component Sec10-like / PH-like domain superfamily / Exocyst complex component EXOC3/Sec6 / Exocyst complex component Sec3, C-terminal / Exocyst complex component Sec3, PIP2-binding N-terminal domain / Exocyst complex component EXOC5/Sec10 / Exocyst complex component EXOC3/Sec6, C-terminal domain / Exocyst component Exo84, C-terminal, subdomain 2 / Exocyst component Exo84, C-terminal / Exocyst component Exo84, C-terminal, subdomain 1 / Exocyst complex component EXOC2/Sec5
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
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 15 Å
AuthorsFrost, A. / Munson, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2R01GM068803 United States
Citation
Journal: J. Cell Biol. / Year: 2020
Title: Exocyst structural changes associated with activation of tethering downstream of Rho/Cdc42 GTPases.
Authors: Guendalina Rossi / Dante Lepore / Lillian Kenner / Alexander B Czuchra / Melissa Plooster / Adam Frost / Mary Munson / Patrick Brennwald /
Abstract: The exocyst complex plays a critical role in determining both temporal and spatial dynamics of exocytic vesicle tethering and fusion with the plasma membrane. However, the mechanism by which the ...The exocyst complex plays a critical role in determining both temporal and spatial dynamics of exocytic vesicle tethering and fusion with the plasma membrane. However, the mechanism by which the exocyst functions and how it is regulated remain poorly understood. Here we describe a novel biochemical assay for the examination of exocyst function in vesicle tethering. Importantly, the assay is stimulated by gain-of-function mutations in the Exo70 component of the exocyst, selected for their ability to bypass Rho/Cdc42 activation in vivo. Single-particle electron microscopy and 3D reconstructions of negatively stained exocyst complexes reveal a structural change in the mutant exocyst that exposes a binding site for the v-SNARE. We demonstrate a v-SNARE requirement in our tethering assay and increased v-SNARE binding to exocyst gain-of-function complexes. Together, these data suggest an allosteric mechanism for activation involving a conformational change in one subunit of the complex, which is relayed through the complex to regulate its biochemical activity in vitro, as well as overall function in vivo.
#1: Journal: 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.
#2: Journal: Nat. Struct. Mol. Biol. / Year: 2016
Title: Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex.
Authors: Margaret R Heider / Mingyu Gu / Caroline M Duffy / Anne M Mirza / Laura L Marcotte / Alexandra C Walls / Nicholas Farrall / Zhanna Hakhverdyan / Mark C Field / Michael P Rout / Adam Frost / Mary Munson /
Abstract: The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified ...The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJan 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release

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Structure visualization

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  • Superimposition on EM map
  • EMDB-21226
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation, assay for oligomerization, microscopy
  • Download structure data
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 / Exocyst / Protein PSL1 / Exocyst


Mass: 154889.547 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P33332
#2: Protein Exocyst complex component SEC5 / Exocyst / Exocyst


Mass: 112236.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P89102
#3: Protein Exocyst complex component SEC6 / Exocyst / Exocyst


Mass: 93539.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32844
#4: Protein Exocyst complex component SEC8 / Exocyst / Exocyst


Mass: 122367.109 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32855
#5: Protein Exocyst complex component SEC10 / Exocyst / Exocyst


Mass: 100459.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q06245
#6: Protein Exocyst complex component SEC15 / Exocyst / Exocyst


Mass: 105166.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P22224
#7: Protein Exocyst complex component EXO70 / Exocyst / Exocyst complex protein of 70 kDa / Exocyst


Mass: 71382.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P19658
#8: Protein Exocyst complex component EXO84 / Exocyst / Exocyst complex protein of 84 kDa / U1 SNP1-associating protein 3 / Exocyst


Mass: 85649.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P38261

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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: NEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: Uranyl Acetate
Specimen supportDetails: unspecified

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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 FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 6466

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Processing

EM softwareName: UCSF Chimera / Category: model fitting
Image processing
IDImage recording-ID
11
21
CTF correction
IDImage processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
116VKLC1 (asymmetric)
226VKL
3D reconstruction

Algorithm: FOURIER SPACE / Entry-ID: 6VKL / Num. of class averages: 1 / Num. of particles: 67509 / Resolution: 15 Å / Resolution method: FSC 0.5 CUT-OFF / Symmetry type: POINT

IDImage processing-ID
11
22
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5YFP
RefinementHighest resolution: 4.4 Å

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