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Open data
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Basic information
Entry | Database: PDB / ID: 8ucq | ||||||
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Title | CryoEM structure of Sec7 autoinhibited conformation | ||||||
![]() | SEC7 domain-containing protein | ||||||
![]() | PROTEIN TRANSPORT / GEF / Exchange Factor / Golgi | ||||||
Function / homology | ![]() regulation of ARF protein signal transduction / guanyl-nucleotide exchange factor activity / protein transport / Golgi apparatus Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å | ||||||
![]() | Brownfield, B.A. / Fromme, J.C. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Sec7 regulatory domains scaffold autoinhibited and active conformations. Authors: Bryce A Brownfield / Brian C Richardson / Steve L Halaby / J Christopher Fromme / ![]() Abstract: The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of ...The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here, we report the cryogenic electron microscopy structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 478.4 KB | Display | ![]() |
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PDB format | ![]() | 360.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 42135MC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 215563.719 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: TT172_LOCUS6419 / Production host: ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Sec7 homodimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Value: 0.39214 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 63000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Average exposure time: 3.092 sec. / Electron dose: 55.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4474 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
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Processing
EM software |
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Image processing | Details: After motion correction, 1,073 images were manually removed leaving 3,401 curated micrographs. | |||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 938642 | |||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196888 / Symmetry type: POINT | |||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL / Details: Phenix Real Space Refinement | |||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | |||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||
Displacement parameters | Biso mean: 81.56 Å2 | |||||||||||||||||||||||||||
Refine LS restraints |
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