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- PDB-6tgc: CryoEM structure of the ternary DOCK2-ELMO1-RAC1 complex -

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Entry
Database: PDB / ID: 6tgc
TitleCryoEM structure of the ternary DOCK2-ELMO1-RAC1 complex
Components
  • Dedicator of cytokinesis protein 2
  • Engulfment and cell motility protein 1ELMO1
  • Ras-related C3 botulinum toxin substrate 1
KeywordsSIGNALING PROTEIN / guanine nucleotide exchange factor / cytoskeleton / actin / cryoEM
Function / homology
Function and homology information


alpha-beta T cell proliferation / membrane raft polarization / myeloid dendritic cell activation involved in immune response / establishment of T cell polarity / guanyl-nucleotide exchange factor complex / regulation of respiratory burst / macropinocytosis / regulation of neutrophil migration / localization within membrane / regulation of hydrogen peroxide metabolic process ...alpha-beta T cell proliferation / membrane raft polarization / myeloid dendritic cell activation involved in immune response / establishment of T cell polarity / guanyl-nucleotide exchange factor complex / regulation of respiratory burst / macropinocytosis / regulation of neutrophil migration / localization within membrane / regulation of hydrogen peroxide metabolic process / Rac guanyl-nucleotide exchange factor activity / negative regulation of interleukin-23 production / engulfment of apoptotic cell / immunological synapse formation / negative regulation of receptor-mediated endocytosis / mast cell chemotaxis / Rac protein signal transduction / Rho GDP-dissociation inhibitor binding / negative thymic T cell selection / ruffle assembly / cortical cytoskeleton organization / cell projection assembly / ruffle organization / regulation of nitric oxide biosynthetic process / small monomeric GTPase / positive thymic T cell selection / positive regulation of neutrophil chemotaxis / hepatocyte growth factor receptor signaling pathway / thioesterase binding / motor neuron axon guidance / regulation of lamellipodium assembly / positive regulation of cell-substrate adhesion / positive regulation of Rho protein signal transduction / positive regulation of lamellipodium assembly / lamellipodium assembly / semaphorin-plexin signaling pathway / regulation of stress fiber assembly / regulation of cell size / small GTPase mediated signal transduction / establishment or maintenance of cell polarity / anatomical structure morphogenesis / ficolin-1-rich granule membrane / positive regulation of microtubule polymerization / positive regulation of focal adhesion assembly / Rho protein signal transduction / regulation of small GTPase mediated signal transduction / Wnt signaling pathway, planar cell polarity pathway / positive regulation of insulin secretion involved in cellular response to glucose stimulus / bone resorption / positive regulation of substrate adhesion-dependent cell spreading / cell motility / Rab GTPase binding / positive regulation of stress fiber assembly / regulation of cell migration / substrate adhesion-dependent cell spreading / regulation of actin cytoskeleton organization / positive regulation of phagocytosis / actin filament polymerization / cell-matrix adhesion / positive regulation of DNA replication / GTPase activator activity / cell projection / T cell receptor binding / secretory granule membrane / actin filament organization / phagocytosis, engulfment / T cell costimulation / ruffle membrane / cytoplasmic ribonucleoprotein granule / neuron migration / ephrin receptor signaling pathway / trans-Golgi network / actin cytoskeleton organization / regulation of defense response to virus by virus / response to wounding / SH3 domain binding / recycling endosome membrane / cell population proliferation / postsynapse / cell cortex / melanosome / histone deacetylase binding / vascular endothelial growth factor receptor signaling pathway / Fc-gamma receptor signaling pathway involved in phagocytosis / specific granule lumen / cellular response to mechanical stimulus / chemotaxis / Fc-epsilon receptor signaling pathway / lamellipodium / cell migration / cytoplasmic vesicle / ATPase binding / regulation of cell shape / dendritic spine / blood coagulation / cytoskeleton / intracellular signal transduction / GTPase activity / cell adhesion / inflammatory response
Small GTP-binding protein domain / DHR-2 domain / Small GTPase Rho / Pleckstrin homology domain / Dedicator of cytokinesis, C-terminal / Small GTPase / Armadillo-like helical / PH-like domain superfamily / Armadillo-type fold / SH3 domain ...Small GTP-binding protein domain / DHR-2 domain / Small GTPase Rho / Pleckstrin homology domain / Dedicator of cytokinesis, C-terminal / Small GTPase / Armadillo-like helical / PH-like domain superfamily / Armadillo-type fold / SH3 domain / Domain of unknown function DUF3361 / Dedicator of cytokinesis / Dedicator of cytokinesis protein 2 / DHR-1 domain / P-loop containing nucleoside triphosphate hydrolase / ELMO domain / Engulfment and cell motility protein 1 / Dedicator of cytokinesis, N-terminal domain / C2 domain superfamily / SH3-like domain superfamily / Dedicator of cytokinesis, N-terminal, subdomain 1 / Dedicator of cytokinesis, C-terminal, lobe A / Dedicator of cytokinesis, C-terminal, lobe C
Ras-related C3 botulinum toxin substrate 1 / Engulfment and cell motility protein 1 / Dedicator of cytokinesis protein 2
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsChang, L. / Yang, J. / Chang, J.H. / Zhang, Z. / Boland, A. / McLaughlin, S.H. / Abu-Thuraia, A. / Killoran, R.C. / Smith, M.J. / Cote, J.F. / Barford, D.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
Marie Sklodowska-Curie Actions, FragNET ITN657725 United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the DOCK2-ELMO1 complex provides insights into regulation of the auto-inhibited state.
Authors: Leifu Chang / Jing Yang / Chang Hwa Jo / Andreas Boland / Ziguo Zhang / Stephen H McLaughlin / Afnan Abu-Thuraia / Ryan C Killoran / Matthew J Smith / Jean-Francois Côté / David Barford /
Abstract: DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCK) ...DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCK) and membrane-associated (DOCK) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMO) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2 complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Dedicator of cytokinesis protein 2
B: Engulfment and cell motility protein 1
C: Ras-related C3 botulinum toxin substrate 1
D: Dedicator of cytokinesis protein 2
E: Engulfment and cell motility protein 1
F: Ras-related C3 botulinum toxin substrate 1


Theoretical massNumber of molelcules
Total (without water)602,5446
Polymers602,5446
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18800 Å2
ΔGint-139 kcal/mol
Surface area210600 Å2
MethodPISA

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Components

#1: Protein Dedicator of cytokinesis protein 2


Mass: 195902.516 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOCK2, KIAA0209 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92608
#2: Protein Engulfment and cell motility protein 1 / ELMO1 / Protein ced-12 homolog / ELMO1


Mass: 83891.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELMO1, KIAA0281 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92556
#3: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 21478.113 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63000, small monomeric GTPase

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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: Ternary complex of DOCK2-ELMO1-RAC1 / Type: COMPLEX / Entity ID: 1, 2, 3 / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesC8H18N2O4S1
2200 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum ER / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
2EPUimage acquisition
4GctfCTF correction
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245763 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 538.5 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.015433882
ELECTRON MICROSCOPYf_angle_d1.789446026
ELECTRON MICROSCOPYf_chiral_restr0.07475416
ELECTRON MICROSCOPYf_plane_restr0.01065930
ELECTRON MICROSCOPYf_dihedral_angle_d15.86320486

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