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- PDB-8zj2: Cryo-EM structure of the RhoG/DOCK5/ELMO1/Rac1 complex -

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Basic information

Entry
Database: PDB / ID: 8zj2
TitleCryo-EM structure of the RhoG/DOCK5/ELMO1/Rac1 complex
Components
  • Dedicator of cytokinesis protein 5
  • Engulfment and cell motility protein 1
  • Ras-related C3 botulinum toxin substrate 1
  • Rho-related GTP-binding protein RhoG
KeywordsSIGNALING PROTEIN / ELMO / DOCK / GEF / GTPASE / RHO / RAC
Function / homology
Function and homology information


regulation of ruffle assembly / negative regulation of vascular associated smooth muscle contraction / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / podosome assembly / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis ...regulation of ruffle assembly / negative regulation of vascular associated smooth muscle contraction / regulation of respiratory burst / regulation of neutrophil migration / negative regulation of interleukin-23 production / localization within membrane / Activated NTRK2 signals through CDK5 / podosome assembly / regulation of hydrogen peroxide metabolic process / negative regulation of receptor-mediated endocytosis / ruffle assembly / NTRK2 activates RAC1 / NADPH oxidase complex / Inactivation of CDC42 and RAC1 / cortical cytoskeleton organization / respiratory burst / guanyl-nucleotide exchange factor complex / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / hepatocyte growth factor receptor signaling pathway / bone remodeling / myoblast fusion / ruffle organization / cell projection assembly / positive regulation of bicellular tight junction assembly / thioesterase binding / negative regulation of fibroblast migration / regulation of stress fiber assembly / RHO GTPases activate CIT / activation of GTPase activity / positive regulation of vascular associated smooth muscle cell migration / regulation of nitric oxide biosynthetic process / regulation of lamellipodium assembly / Nef and signal transduction / sphingosine-1-phosphate receptor signaling pathway / motor neuron axon guidance / RHO GTPases activate KTN1 / PCP/CE pathway / Activation of RAC1 / MET activates RAP1 and RAC1 / DCC mediated attractive signaling / Azathioprine ADME / positive regulation of cell-substrate adhesion / positive regulation of neutrophil chemotaxis / Sema4D mediated inhibition of cell attachment and migration / Ephrin signaling / CD28 dependent Vav1 pathway / Wnt signaling pathway, planar cell polarity pathway / lamellipodium assembly / podosome / anchoring junction / small GTPase-mediated signal transduction / Activation of RAC1 downstream of NMDARs / regulation of cell size / phagocytosis, engulfment / positive regulation of Rho protein signal transduction / NRAGE signals death through JNK / establishment or maintenance of cell polarity / Rho GDP-dissociation inhibitor binding / Rac protein signal transduction / RHO GTPases activate PAKs / semaphorin-plexin signaling pathway / regulation of postsynapse assembly / ficolin-1-rich granule membrane / positive regulation of epithelial cell migration / RHOG GTPase cycle / Sema3A PAK dependent Axon repulsion / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate NADPH Oxidases / anatomical structure morphogenesis / positive regulation of focal adhesion assembly / Rho protein signal transduction / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / membrane scission GTPase motor activity / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / RHO GTPases activate PKNs / positive regulation of lamellipodium assembly / GPVI-mediated activation cascade / positive regulation of stress fiber assembly / positive regulation of microtubule polymerization / EPHB-mediated forward signaling / positive regulation of substrate adhesion-dependent cell spreading / RAC1 GTPase cycle / actin filament polymerization / positive regulation of endothelial cell migration / substrate adhesion-dependent cell spreading / GTPase activator activity / regulation of cell migration / actin filament organization / secretory granule membrane / guanyl-nucleotide exchange factor activity / small monomeric GTPase / cell-matrix adhesion / cell projection / Signal transduction by L1 / VEGFR2 mediated vascular permeability / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of protein localization to plasma membrane / cell chemotaxis
Similarity search - Function
Rho-related GTP-binding protein RhoG / Dedicator of cytokinesis protein 5 / : / Dedicator of cytokinesis, N-terminal domain / Dedicator of cytokinesis, N-terminal, subdomain 1 / DOCK N-terminus / ELMO domain / : / ELMO/CED-12 family / ELMO domain profile. ...Rho-related GTP-binding protein RhoG / Dedicator of cytokinesis protein 5 / : / Dedicator of cytokinesis, N-terminal domain / Dedicator of cytokinesis, N-terminal, subdomain 1 / DOCK N-terminus / ELMO domain / : / ELMO/CED-12 family / ELMO domain profile. / ELMO, armadillo-like helical domain / ELMO, armadillo-like helical domain / Dedicator of cytokinesis / C2 DOCK-type domain / DOCKER domain / Dedicator of cytokinesis, C-terminal, lobe A / Dedicator of cytokinesis, C-terminal, lobe C / DOCKER, Lobe A / DOCKER, Lobe B / DOCKER, Lobe C / DHR-2, Lobe A / C2 domain in Dock180 and Zizimin proteins / DHR-2, Lobe C / DHR-2, Lobe B / C2 DOCK-type domain profile. / DOCKER domain profile. / Pleckstrin homology domain / Small GTPase Rho / small GTPase Rho family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / C2 domain superfamily / Pleckstrin homology domain / SH3 domain / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Armadillo-like helical / Small GTP-binding protein domain / PH-like domain superfamily / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Ras-related C3 botulinum toxin substrate 1 / Rho-related GTP-binding protein RhoG / Engulfment and cell motility protein 1 / Dedicator of cytokinesis protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.66 Å
AuthorsKukimoto-Niino, M. / Katsura, K. / Ishizuka-Katsura, Y. / Mishima-Tsumagari, C. / Yonemochi, M. / Inoue, M. / Nakagawa, R. / Kaushik, R. / Zhang, K.Y.J. / Shirouzu, M.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)KAKENHI JP19K06575 Japan
Japan Society for the Promotion of Science (JSPS)KAKENHI JP22KH05551 Japan
Japan Science and TechnologyCREST JPMJCR22E3 Japan
CitationJournal: J Biol Chem / Year: 2024
Title: RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.
Authors: Mutsuko Kukimoto-Niino / Kazushige Katsura / Yoshiko Ishizuka-Katsura / Chiemi Mishima-Tsumagari / Mayumi Yonemochi / Mio Inoue / Reiko Nakagawa / Rahul Kaushik / Kam Y J Zhang / Mikako Shirouzu /
Abstract: The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK- ...The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
History
DepositionMay 14, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Dedicator of cytokinesis protein 5
C: Ras-related C3 botulinum toxin substrate 1
F: Dedicator of cytokinesis protein 5
G: Ras-related C3 botulinum toxin substrate 1
E: Engulfment and cell motility protein 1
A: Engulfment and cell motility protein 1
D: Rho-related GTP-binding protein RhoG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)615,0589
Polymers614,5117
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 7 molecules BFCGEAD

#1: Protein Dedicator of cytokinesis protein 5


Mass: 191492.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOCK5 / Production host: Homo sapiens (human) / References: UniProt: Q9H7D0
#2: Protein Ras-related C3 botulinum toxin substrate 1 / Cell migration-inducing gene 5 protein / Ras-like protein TC25 / p21-Rac1


Mass: 20244.258 Da / Num. of mol.: 2 / Mutation: G15A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Escherichia coli (E. coli) / References: UniProt: P63000, small monomeric GTPase
#3: Protein Engulfment and cell motility protein 1 / Protein ced-12 homolog


Mass: 84337.719 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELMO1, KIAA0281 / Production host: Homo sapiens (human) / References: UniProt: Q92556
#4: Protein Rho-related GTP-binding protein RhoG


Mass: 22362.359 Da / Num. of mol.: 1 / Mutation: Q61L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOG, ARHG / Production host: Escherichia coli (E. coli) / References: UniProt: P84095

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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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: RhoG/DOCK5/ELMO1/Rac1 complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding 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 IV / 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 FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11976

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2483502
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169096 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17DPA

7dpa

17DPA1PDBexperimental model
26IE1

6ie1

16IE12PDBexperimental model
37Y4A

7y4a

17Y4A3PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00639399
ELECTRON MICROSCOPYf_angle_d0.9353235
ELECTRON MICROSCOPYf_dihedral_angle_d9.5225171
ELECTRON MICROSCOPYf_chiral_restr0.0525913
ELECTRON MICROSCOPYf_plane_restr0.0066816

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