Evidence: homology, 19F NMR shows RhoA and RhoGAP forms 1:1 complex
Type
Name
Symmetry operation
Number
identity operation
1_555
x,y,z
1
Buried area
4240 Å2
ΔGint
-41 kcal/mol
Surface area
16420 Å2
Method
PISA
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 number
20
Space group name H-M
C2221
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Components
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Protein , 2 types, 2 molecules AB
#1: Protein
RhoGTPase-activatingprotein1 / 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
TransformingproteinRhoA / 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
Mass: 18.015 Da / Num. of mol.: 254 / Source method: isolated from a natural source / Formula: H2O
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Details
Sequence details
THE GAP MOTIF FROM RESIDUE 198 TO 439 IS USED FOR CRYSTALLISATION.
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.4 Å3/Da / Density % sol: 49 %
Crystal grow
Temperature: 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
Diffraction
Mean temperature: 100 K / Serial crystal experiment: N
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.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.20681
2407
5 %
RANDOM
Rwork
0.17694
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obs
0.17849
45459
99.94 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å