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- PDB-6bbq: Model for extended volume of truncated monomeric Cytohesin-3 (Grp... -

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Entry
Database: PDB / ID: 6bbq
TitleModel for extended volume of truncated monomeric Cytohesin-3 (Grp1; amino acids 63-399) E161A Arf6 Q67L fusion protein
ComponentsCytohesin-3,ADP-ribosylation factor 6
KeywordsLIPID BINDING PROTEIN / Guanine nucleotide exchange factor / Arf GTPase / Fusion protein / Inositol 1 / 3 / 4 / 5-tetrakisphosphate
Function/homologymyeloid cell apoptotic process / Intra-Golgi traffic / protein localization to endosome / regulation of dendritic spine development / Sec7 domain superfamily / regulation of ARF protein signal transduction / Sec7 domain / SEC7 domain profile. / Sec7, C-terminal domain superfamily / Golgi vesicle transport ...myeloid cell apoptotic process / Intra-Golgi traffic / protein localization to endosome / regulation of dendritic spine development / Sec7 domain superfamily / regulation of ARF protein signal transduction / Sec7 domain / SEC7 domain profile. / Sec7, C-terminal domain superfamily / Golgi vesicle transport / establishment of epithelial cell polarity / regulation of Rac protein signal transduction / negative regulation of receptor-mediated endocytosis / negative regulation of dendrite development / Sec7 domain / ARF guanyl-nucleotide exchange factor activity / regulation of filopodium assembly / small GTPase Arf family profile. / Small GTPase superfamily, ARF type / Flemming body / Small GTPase superfamily, ARF/SAR type / ruffle organization / protein localization to cell surface / thioesterase binding / filopodium membrane / ADP-ribosylation factor family / MET receptor recycling / TBC/RABGAPs / cortical actin cytoskeleton organization / phosphatidylinositol-3,4,5-trisphosphate binding / cleavage furrow / hepatocyte apoptotic process / positive regulation of cell adhesion / positive regulation of actin filament polymerization / adherens junction / small GTPase mediated signal transduction / guanyl-nucleotide exchange factor activity / bicellular tight junction / vesicle-mediated transport / endocytic vesicle / ruffle / liver development / movement of cell or subcellular component / positive regulation of protein localization to plasma membrane / PH domain / PH domain profile. / Pleckstrin homology domain / nervous system development / recycling endosome membrane / cell cortex / Clathrin-mediated endocytosis / protein transport / extrinsic component of cytoplasmic side of plasma membrane / protein N-terminus binding / Small GTP-binding protein domain / early endosome / cell differentiation / PH-like domain superfamily / endosome / GTPase activity / cell cycle / cell division / cell adhesion / myelin sheath / GTP binding / focal adhesion / Golgi apparatus / P-loop containing nucleoside triphosphate hydrolase / membrane / extracellular exosome / nucleoplasm / plasma membrane / cytosol / cytoplasm / Cytohesin-3 / ADP-ribosylation factor 6
Function and homology information
Specimen sourceMus musculus / House mouse / mammal /
Homo sapiens / / human
MethodElectron microscopy (35 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsDas, S. / Malaby, A.W. / Lambright, D.G.
CitationJournal: Structure / Year: 2018
Title: Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors.
Authors: Andrew W Malaby / Sanchaita Das / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / David G Lambright
Abstract: Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and ...Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.
Copyright: 2017 Elsevier Ltd. All rights reserved.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 19, 2017 / Release: Jan 10, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 10, 2018Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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Assembly

Deposited unit
A: Cytohesin-3,ADP-ribosylation factor 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,3554
Polyers60,3081
Non-polymers1,0483
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Cytohesin-3,ADP-ribosylation factor 6 / ARF nucleotide-binding site opener 3 / Protein ARNO3 / General receptor of phosphoinositides 1 / Grp1 / PH / SEC7 and coiled-coil domain-containing protein 3 / CLM3 / SEC7 homolog C / mSec7-3


Mass: 60307.855 Da / Num. of mol.: 1 / Mutation: E161A,E161A
Source: (gene. exp.) Mus musculus / House mouse / mammal / , (gene. exp.) Homo sapiens / / human
Gene: Cyth3, Grp1, Pscd3, ARF6 / Production host: Escherichia coli / References: UniProt:O08967, UniProt:P62330
#2: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3 / : Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / : Magnesium
#4: Chemical ChemComp-4IP / INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE


Mass: 500.075 Da / Num. of mol.: 1 / Formula: C6H16O18P4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Truncated monomeric Cytohesin-3 (Grp1; amino acids 63-399) E161A 6GS Arf6 Q67L fusion protein
Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus
Source (recombinant)Organism: Escherichia coli
Buffer solutionDetails: 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 0.1% 2-mercaptoethanol, and 0.001 mM IP4
pH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Details: Stained with 0.75% (w/v) uranyl formate / Material: Uranyl Formate
Specimen supportGrid material: COPPER / Grid mesh size: 400

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TECNAI 12
Details: Gatan Erlang Shen 785 camera used for collecting images
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 / Nominal defocus max: 3200 nm / Nominal defocus min: 1200 nm / Cs: 2 mm
Image recordingElectron dose: 20 e/Å2
Details: Gatan Erlang Shen 785 camera used for collecting images
Film or detector model: OTHER / Number of grids imaged: 1 / Number of real images: 369

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Processing

EM software
IDNameVersionCategory
4EMAN2CTF CORRECTION
12EMAN2CLASSIFICATION
13EMAN2RECONSTRUCTION
Image processingDetails: The images were X-ray corrected
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: EMAN2 based manual particle picking / Number of particles selected: 10000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 35 Å / Resolution method: FSC 0.5 CUT-OFF / Number of particles: 6504 / Number of class averages: 71 / Symmetry type: POINT

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