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- PDB-6u3g: Best fitting antiparallel model for Volume 2 of truncated dimeric... -

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Basic information

Entry
Database: PDB / ID: 6u3g
TitleBest fitting antiparallel model for Volume 2 of truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399)
ComponentsCytohesin-3
KeywordsENDOCYTOSIS / Arf GEF / Phosphoinositide binding / Sec7 Domain / PH Domain
Function / homology
Function and homology information


Intra-Golgi traffic / regulation of ARF protein signal transduction / Golgi vesicle transport / establishment of epithelial cell polarity / phosphatidylinositol-3,4,5-trisphosphate binding / bicellular tight junction / positive regulation of cell adhesion / ruffle / guanyl-nucleotide exchange factor activity / adherens junction ...Intra-Golgi traffic / regulation of ARF protein signal transduction / Golgi vesicle transport / establishment of epithelial cell polarity / phosphatidylinositol-3,4,5-trisphosphate binding / bicellular tight junction / positive regulation of cell adhesion / ruffle / guanyl-nucleotide exchange factor activity / adherens junction / extrinsic component of cytoplasmic side of plasma membrane / Golgi membrane / nucleoplasm / plasma membrane / cytosol
Similarity search - Function
Sec7 domain / SEC7 domain profile. / Sec7 domain / Sec7 domain superfamily / Sec7, C-terminal domain superfamily / Sec7 domain / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / PH-like domain superfamily
Similarity search - Domain/homology
INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE / Cytohesin-3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 53 Å
AuthorsDas, S. / Lambright, D.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM056324 United States
CitationJournal: Structure / Year: 2019
Title: Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.
Authors: Sanchaita Das / Andrew W Malaby / Agata Nawrotek / Wenhua Zhang / Mahel Zeghouf / Sarah Maslen / Mark Skehel / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / Jacqueline Cherfils / ...Authors: Sanchaita Das / Andrew W Malaby / Agata Nawrotek / Wenhua Zhang / Mahel Zeghouf / Sarah Maslen / Mark Skehel / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / Jacqueline Cherfils / David G Lambright /
Abstract: Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through ...Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.
History
DepositionAug 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Dec 18, 2019Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Cytohesin-3
B: Cytohesin-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,0044
Polymers93,0042
Non-polymers1,0002
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, equilibrium centrifugation, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3970 Å2
ΔGint-26 kcal/mol
Surface area45070 Å2

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Components

#1: Protein Cytohesin-3 / ARF nucleotide-binding site opener 3 / Protein ARNO3 / General receptor of phosphoinositides 1 / ...ARF nucleotide-binding site opener 3 / Protein ARNO3 / General receptor of phosphoinositides 1 / Grp1 / PH / SEC7 and coiled-coil domain-containing protein 3


Mass: 46501.766 Da / Num. of mol.: 2 / Fragment: UNP residues 13-400
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CYTH3, ARNO3, GRP1, PSCD3 / Production host: Escherichia coli BL21(DE3) (unknown) / References: UniProt: O43739
#2: Chemical ChemComp-4IP / INOSITOL-(1,3,4,5)-TETRAKISPHOSPHATE


Mass: 500.075 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H16O18P4
Has ligand of interestN

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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: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (unknown) / Strain: BL21(DE3)
Buffer solutionpH: 8
Details: Peak fractions after gel filtration were immediately diluted, applied to freshly glow discharged grids, and stained with uranyl formate.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: The sample is a uniform homodimer with significant conformational flexibility.
EM stainingType: NEGATIVE / Details: Stained with 0.75% (w/v) uranyl formate / Material: Uranyl Formate
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in.

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM120T
Details: specimen holder: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 28000 X / Nominal defocus max: -3200 nm / Nominal defocus min: -1200 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Num. of grids imaged: 1 / Num. of real images: 500
Image scansWidth: 2048 / Height: 2048

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN22.12particle selectionParticles were manually picked
2SerialEMimage acquisition
4EMAN22.12CTF correction
7ADP_EM0.99model fitting
12EMAN22.12classification
13EMAN22.123D reconstruction
Image processingDetails: Images were processed with EMAN2 after X-ray removal with IMOD
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6504 / Details: Particles were manually picked.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 53 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 1863 / Algorithm: BACK PROJECTION / Num. of class averages: 15 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: The model with the best correlation coefficient was selected by ADP_EM from a pool of 10000 models generated by RRT_SAMPLE using rigid bodies derived from 2R09 (autoinhibited core, amino ...Details: The model with the best correlation coefficient was selected by ADP_EM from a pool of 10000 models generated by RRT_SAMPLE using rigid bodies derived from 2R09 (autoinhibited core, amino acids 63-399) and a canonical antiparallel coiled coil (amino acids 18-53) built with CCBuilder. Flexible regions with reasonable geometry were modeled with MODELLER.
Atomic model buildingPDB-ID: 2R09
Pdb chain-ID: B / Pdb chain residue range: 63-399

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