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- EMDB-21226: Negative stain reconstruction of the yeast exocyst octameric complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-21226
TitleNegative stain reconstruction of the yeast exocyst octameric complex.
Map data
SampleNEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX
  • (Exocyst complex component ...Exocyst) x 8
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) / Baker's yeast (baker's yeast)
Methodsingle particle reconstruction / Resolution: 15 Å
AuthorsFrost A / Munson M
Funding support United States, 1 items
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 ReportPDB-ID: 6vkl

SummaryFull reportAbout validation report
History
DepositionJan 21, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6vkl
  • Surface level: 4.1
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21226.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.87 Å/pix.
x 256 pix.
= 734.976 Å
2.87 Å/pix.
x 256 pix.
= 734.976 Å
2.87 Å/pix.
x 256 pix.
= 734.976 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.871 Å
Density
Contour LevelBy AUTHOR: 4.1 / Movie #1: 4.1
Minimum - Maximum-2.171029 - 11.401691
Average (Standard dev.)0.028356869 (±0.40476105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 734.976 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.8712.8712.871
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z734.976734.976734.976
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.17111.4020.028

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Supplemental data

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Sample components

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Entire NEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX

EntireName: NEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX / Number of components: 9

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Component #1: protein, NEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX

ProteinName: NEGATIVE STAIN MAP OF THE YEAST EXOCYST COMPLEX / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Exocyst complex component SEC3

ProteinName: Exocyst complex component SEC3Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 154.889547 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #3: protein, Exocyst complex component SEC5

ProteinName: Exocyst complex component SEC5Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 112.236875 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #4: protein, Exocyst complex component SEC6

ProteinName: Exocyst complex component SEC6Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 93.539703 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #5: protein, Exocyst complex component SEC8

ProteinName: Exocyst complex component SEC8Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 122.367109 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #6: protein, Exocyst complex component SEC10

ProteinName: Exocyst complex component SEC10Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 100.459578 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #7: protein, Exocyst complex component SEC15

ProteinName: Exocyst complex component SEC15Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 105.166641 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #8: protein, Exocyst complex component EXO70

ProteinName: Exocyst complex component EXO70Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 71.382328 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #9: protein, Exocyst complex component EXO84

ProteinName: Exocyst complex component EXO84Exocyst / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 85.649672 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle
Sample solutionSpecimen conc.: 0.3 mg/mL / pH: 7.4
Support filmunspecified
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000.0 - 3000.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 6466

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 67509
3D reconstruction #1Algorithm: FOURIER SPACE / Resolution: 15 Å / Resolution method: FSC 0.5 CUT-OFF
3D reconstruction #2Algorithm: FOURIER SPACE / Resolution: 15 Å / Resolution method: FSC 0.5 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 5YFP
Output model

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