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- PDB-8ucq: CryoEM structure of Sec7 autoinhibited conformation -

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Basic information

Entry
Database: PDB / ID: 8ucq
TitleCryoEM structure of Sec7 autoinhibited conformation
ComponentsSEC7 domain-containing protein
KeywordsPROTEIN TRANSPORT / GEF / Exchange Factor / Golgi
Function / homology
Function and homology information


regulation of ARF protein signal transduction / guanyl-nucleotide exchange factor activity / protein transport / Golgi apparatus
Similarity search - Function
Sec7/BIG1-like, C-terminal domain / BIG2 C-terminal domain / Mon2/Sec7/BIG1-like, HDS / Mon2/Sec7/BIG1-like, HDS / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily ...Sec7/BIG1-like, C-terminal domain / BIG2 C-terminal domain / Mon2/Sec7/BIG1-like, HDS / Mon2/Sec7/BIG1-like, HDS / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / Armadillo-type fold
Similarity search - Domain/homology
A7bd1859-5d22-4ce8-aff3-d8d273e6acf0
Similarity search - Component
Biological speciesThermothielavioides terrestris (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBrownfield, B.A. / Fromme, J.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136258 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
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
DepositionSep 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: SEC7 domain-containing protein
A: SEC7 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)431,1272
Polymers431,1272
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein SEC7 domain-containing protein


Mass: 215563.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermothielavioides terrestris (fungus)
Gene: TT172_LOCUS6419 / Production host: Komagataella pastoris (fungus) / References: UniProt: A0A3S5CXE1

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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: Sec7 homodimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.39214 MDa / Experimental value: NO
Source (natural)Organism: Thermothielavioides terrestris (fungus)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes pH 7.4C8H18N2O4S1
2250 mMSodium chlorideNaClSodium chloride1
32 mMDithiothreitolC4H10O2S21
41 mMFOS-Choline-8, FluorinatedC13H17F13NO4P1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: 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
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 63000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIXmodel refinement
Image processingDetails: After motion correction, 1,073 images were manually removed leaving 3,401 curated micrographs.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 938642
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196888 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: Phenix Real Space Refinement
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 81.56 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003120144
ELECTRON MICROSCOPYf_angle_d0.56827180
ELECTRON MICROSCOPYf_chiral_restr0.03743164
ELECTRON MICROSCOPYf_plane_restr0.00293410
ELECTRON MICROSCOPYf_dihedral_angle_d3.93572634

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