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- PDB-6r3v: Crystal Structure of RhoA-GDP-Pi in Complex with RhoGAP -

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Basic information

Entry
Database: PDB / ID: 6r3v
TitleCrystal Structure of RhoA-GDP-Pi in Complex with RhoGAP
Components
  • Rho GTPase-activating protein 1
  • Transforming protein RhoA
KeywordsHYDROLASE / SMALL G PROTEIN / GTPASE / GTP HYDROLYSIS / PRODUCT COMPLEX
Function / homology
Function and homology information


negative regulation of endocytic recycling / transferrin transport / aortic valve formation / alpha-beta T cell lineage commitment / mitotic cleavage furrow formation / bone trabecula morphogenesis / positive regulation of lipase activity / endothelial tube lumen extension / skeletal muscle satellite cell migration / positive regulation of vascular associated smooth muscle contraction ...negative regulation of endocytic recycling / transferrin transport / aortic valve formation / alpha-beta T cell lineage commitment / mitotic cleavage furrow formation / bone trabecula morphogenesis / positive regulation of lipase activity / endothelial tube lumen extension / skeletal muscle satellite cell migration / positive regulation of vascular associated smooth muscle contraction / angiotensin-mediated vasoconstriction involved in regulation of systemic arterial blood pressure / SLIT2:ROBO1 increases RHOA activity / RHO GTPases Activate Rhotekin and Rhophilins / Roundabout signaling pathway / negative regulation of intracellular steroid hormone receptor signaling pathway / Axonal growth inhibition (RHOA activation) / Axonal growth stimulation / regulation of neural precursor cell proliferation / cleavage furrow formation / regulation of modification of postsynaptic actin cytoskeleton / regulation of osteoblast proliferation / forebrain radial glial cell differentiation / cell junction assembly / apical junction assembly / regulation of systemic arterial blood pressure by endothelin / cellular response to chemokine / negative regulation of cell migration involved in sprouting angiogenesis / beta selection / establishment of epithelial cell apical/basal polarity / regulation of modification of postsynaptic structure / negative regulation of cell size / RHO GTPases Activate ROCKs / negative regulation of oxidative phosphorylation / negative regulation of motor neuron apoptotic process / RHOF GTPase cycle / RHO GTPases activate CIT / RHOD GTPase cycle / PCP/CE pathway / Sema4D induced cell migration and growth-cone collapse / RHO GTPases activate KTN1 / apolipoprotein A-I-mediated signaling pathway / positive regulation of podosome assembly / negative regulation of cell-substrate adhesion / Wnt signaling pathway, planar cell polarity pathway / regulation of small GTPase mediated signal transduction / Sema4D mediated inhibition of cell attachment and migration / ossification involved in bone maturation / positive regulation of alpha-beta T cell differentiation / odontogenesis / motor neuron apoptotic process / wound healing, spreading of cells / RND2 GTPase cycle / PI3K/AKT activation / positive regulation of leukocyte adhesion to vascular endothelial cell / endosomal transport / apical junction complex / regulation of focal adhesion assembly / negative chemotaxis / myosin binding / RHOB GTPase cycle / small GTPase-mediated signal transduction / EPHA-mediated growth cone collapse / stress fiber assembly / regulation of neuron projection development / RHOJ GTPase cycle / RHOC GTPase cycle / RHOQ GTPase cycle / androgen receptor signaling pathway / positive regulation of cytokinesis / cellular response to cytokine stimulus / cerebral cortex cell migration / ERBB2 Regulates Cell Motility / cleavage furrow / semaphorin-plexin signaling pathway / CDC42 GTPase cycle / Rho protein signal transduction / ficolin-1-rich granule membrane / RHOG GTPase cycle / mitotic spindle assembly / RHOA GTPase cycle / endothelial cell migration / RAC3 GTPase cycle / RAC2 GTPase cycle / positive regulation of T cell migration / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / regulation of microtubule cytoskeleton organization / cytoplasmic microtubule organization / skeletal muscle tissue development / regulation of cell migration / negative regulation of reactive oxygen species biosynthetic process / RHO GTPases activate PKNs / positive regulation of stress fiber assembly / GPVI-mediated activation cascade / RAC1 GTPase cycle / EPHB-mediated forward signaling / substantia nigra development / positive regulation of neuron differentiation / GTPase activator activity / substrate adhesion-dependent cell spreading / cell-matrix adhesion
Similarity search - Function
: / Phosphatidylinositol 3-kinase; Chain A / Rho GTPase activation protein / Divergent CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) / CRAL-TRIO lipid binding domain / CRAL-TRIO lipid binding domain superfamily / Rho GTPase-activating protein domain / RhoGAP domain ...: / Phosphatidylinositol 3-kinase; Chain A / Rho GTPase activation protein / Divergent CRAL/TRIO domain / CRAL-TRIO lipid binding domain profile. / Domain in homologues of a S. cerevisiae phosphatidylinositol transfer protein (Sec14p) / CRAL-TRIO lipid binding domain / CRAL-TRIO lipid binding domain superfamily / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / Small GTPase Rho / small GTPase Rho family profile. / Rho GTPase activation protein / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
2,3-DIHYDROXY-1,4-DITHIOBUTANE / GUANOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Transforming protein RhoA / Rho GTPase-activating protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsJin, Y.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust209057/Z/17/Z United Kingdom
CitationJournal: Chemistry / Year: 2019
Title: A GAP-GTPase-GDP-PiIntermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers.
Authors: Molt Jr., R.W. / Pellegrini, E. / Jin, Y.
History
DepositionMar 21, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2Jul 17, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Rho GTPase-activating protein 1
B: Transforming protein RhoA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,3028
Polymers72,2952
Non-polymers1,0076
Water4,576254
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: homology, 19F NMR shows RhoA and RhoGAP forms 1:1 complex
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4240 Å2
ΔGint-41 kcal/mol
Surface area16420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)114.060, 122.780, 67.190
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Rho GTPase-activating protein 1 / CDC42 GTPase-activating protein / GTPase-activating protein rhoGAP / Rho-related small GTPase ...CDC42 GTPase-activating protein / GTPase-activating protein rhoGAP / Rho-related small GTPase protein activator / Rho-type GTPase-activating protein 1 / p50-RhoGAP


Mass: 50495.508 Da / Num. of mol.: 1 / Fragment: GTPASE ACTIVITING DOMAIN
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARHGAP1, CDC42GAP, RHOGAP1 / Plasmid: PGEX2T / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): ROSETTA PLYSS / References: UniProt: Q07960
#2: Protein Transforming protein RhoA / / Rho cDNA clone 12 / h12


Mass: 21799.158 Da / Num. of mol.: 1 / Mutation: YES [F25N]
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RHOA, ARH12, ARHA, RHO12 / Plasmid: PGEX2T / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): ROSETTA PLYSS / References: UniProt: P61586

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Non-polymers , 6 types, 260 molecules

#3: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID / MES (buffer)


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL / Dithiothreitol


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 254 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHE GAP MOTIF FROM RESIDUE 198 TO 439 IS USED FOR CRYSTALLISATION.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.4 Å3/Da / Density % sol: 49 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: Equal amounts of reservoir solution containing 18% PEG2000 MME, 100 mM MES, pH 6.0, 10 mM MgCl2, 3 mM DTT, 3 mM NaN3, 120 mM ammonium sulphate and stock solution of the RhoA/RhoGAP protein ...Details: Equal amounts of reservoir solution containing 18% PEG2000 MME, 100 mM MES, pH 6.0, 10 mM MgCl2, 3 mM DTT, 3 mM NaN3, 120 mM ammonium sulphate and stock solution of the RhoA/RhoGAP protein complex at 800 mM in 20 mM Tris.HCl, pH 7.4, with 2 mM AlCl3, 10 mM MgCl2, and 20 mM NaF.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.91732 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: May 24, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91732 Å / Relative weight: 1
ReflectionResolution: 1.75→43.48 Å / Num. obs: 47885 / % possible obs: 100 % / Observed criterion σ(I): 1.4 / Redundancy: 6.7 % / Rmerge(I) obs: 0.04 / Net I/σ(I): 19.1
Reflection shellResolution: 1.75→1.78 Å / Redundancy: 6.7 % / Rmerge(I) obs: 1.3 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 2635 / CC1/2: 0.679 / Rpim(I) all: 1.56 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0131refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: WWPDB ENTRY 1OXT

1oxt


Resolution: 1.75→43.48 Å / Cor.coef. Fo:Fc: 0.971 / Cor.coef. Fo:Fc free: 0.962 / SU B: 2.872 / SU ML: 0.086 / Cross valid method: THROUGHOUT / ESU R: 0.099 / ESU R Free: 0.098
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. RESIDUES 25-33 IN CHAIN B ARE DISORDERED.
RfactorNum. reflection% reflectionSelection details
Rfree0.20681 2407 5 %RANDOM
Rwork0.17694 ---
obs0.17849 45459 99.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 38.633 Å2
Baniso -1Baniso -2Baniso -3
1-1.99 Å20 Å2-0 Å2
2--0.43 Å20 Å2
3----2.42 Å2
Refinement stepCycle: LAST / Resolution: 1.75→43.48 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2927 0 59 254 3240
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0193053
X-RAY DIFFRACTIONr_bond_other_d0.0020.022925
X-RAY DIFFRACTIONr_angle_refined_deg1.7611.9914154
X-RAY DIFFRACTIONr_angle_other_deg1.02236724
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5055367
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.8124.857140
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.07215505
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.4351517
X-RAY DIFFRACTIONr_chiral_restr0.1060.2476
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0213388
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02669
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.8363.671477
X-RAY DIFFRACTIONr_mcbond_other2.8363.6671476
X-RAY DIFFRACTIONr_mcangle_it3.8485.4831841
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it3.5984.0231576
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.75→1.795 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.32 175 -
Rwork0.317 3330 -
obs--99.8 %

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