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- PDB-4fss: Crystal structure of a RAS p21 protein activator (RASA1) from Hom... -

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Basic information

Entry
Database: PDB / ID: 4fss
TitleCrystal structure of a RAS p21 protein activator (RASA1) from Homo sapiens at 2.25 A resolution
ComponentsRas GTPase-activating protein 1
KeywordsSIGNALING PROTEIN / GTPase activating protein / SH3 domain / RAS signaling pathway / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-BIOLOGY / Partnership for T-Cell Biology / TCELL
Function / homology
Function and homology information


regulation of RNA metabolic process / regulation of actin filament polymerization / potassium channel inhibitor activity / negative regulation of cell adhesion / blood vessel morphogenesis / negative regulation of cell-matrix adhesion / mitotic cytokinesis / ephrin receptor signaling pathway / vasculogenesis / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases ...regulation of RNA metabolic process / regulation of actin filament polymerization / potassium channel inhibitor activity / negative regulation of cell adhesion / blood vessel morphogenesis / negative regulation of cell-matrix adhesion / mitotic cytokinesis / ephrin receptor signaling pathway / vasculogenesis / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / ruffle / EPHB-mediated forward signaling / phosphotyrosine residue binding / GTPase activator activity / Downstream signal transduction / VEGFR2 mediated cell proliferation / Regulation of RAS by GAPs / GTPase binding / regulation of cell shape / negative regulation of neuron apoptotic process / intracellular signal transduction / signaling receptor binding / GTPase activity / negative regulation of apoptotic process / signal transduction / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ras GTPase-activating protein 1, N-terminal SH2 domain / RasGAP, SH3 domain / Ras GTPase-activating protein 1, C-terminal SH2 domain / Ras GTPase-activating protein / Ras GTPase-activating protein, conserved site / Ras GTPase-activating proteins domain signature. / GTPase-activator protein for Ras-like GTPase / Ras GTPase-activating proteins profile. / GTPase-activator protein for Ras-like GTPases / Ras GTPase-activating domain ...Ras GTPase-activating protein 1, N-terminal SH2 domain / RasGAP, SH3 domain / Ras GTPase-activating protein 1, C-terminal SH2 domain / Ras GTPase-activating protein / Ras GTPase-activating protein, conserved site / Ras GTPase-activating proteins domain signature. / GTPase-activator protein for Ras-like GTPase / Ras GTPase-activating proteins profile. / GTPase-activator protein for Ras-like GTPases / Ras GTPase-activating domain / Rho GTPase activation protein / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / SH3 Domains / PH domain / C2 domain superfamily / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / Src homology 3 domains / SH2 domain / SH3 type barrels. / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / PH-like domain superfamily / Roll / Mainly Beta
Similarity search - Domain/homology
PHOSPHATE ION / Ras GTPase-activating protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.25 Å
AuthorsJoint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
CitationJournal: To be published
Title: Crystal structure of a RAS p21 protein activator (RASA1) from Homo sapiens at 2.25 A resolution
Authors: Joint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
History
DepositionJun 27, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 18, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2015Group: Database references / Structure summary
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software
Revision 1.3Feb 1, 2023Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ras GTPase-activating protein 1
B: Ras GTPase-activating protein 1
C: Ras GTPase-activating protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,3798
Polymers22,0863
Non-polymers2935
Water1,27971
1
A: Ras GTPase-activating protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,4923
Polymers7,3621
Non-polymers1302
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Ras GTPase-activating protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,3972
Polymers7,3621
Non-polymers351
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Ras GTPase-activating protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,4903
Polymers7,3621
Non-polymers1282
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)82.220, 82.220, 157.212
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
DetailsCRYSTAL PACKING ANALYSIS SUGGESTS THE ASSIGNMENT OF A MONOMER AS THE SIGNIFICANT OLIGOMERIZATION STATE.

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Components

#1: Protein Ras GTPase-activating protein 1 / GAP / GTPase-activating protein / RasGAP / Ras p21 protein activator / p120GAP


Mass: 7362.018 Da / Num. of mol.: 3 / Fragment: SH3 domain residues 281-341
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BC033015, RASA, RASA1 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: P20936
#2: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 71 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 281-341 OF THE TARGET SEQUENCE.

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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: 3.47 Å3/Da / Density % sol: 64.58 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 20.00% Glycerol, 1.600M NH4H2PO4, 0.1M TRIS pH 8.5, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL11-1 / Wavelength: 0.91837,0.97968,0.97901
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 8, 2012
Details: Flat mirror (vertical focusing), single crystal Si(111) bent monochromator (horizontal focusing)
RadiationMonochromator: single crystal Si(111) bent / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.918371
20.979681
30.979011
ReflectionResolution: 2.25→29.449 Å / Num. obs: 15649 / % possible obs: 99.7 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 36.791 Å2 / Rmerge(I) obs: 0.223 / Net I/σ(I): 7.78
Reflection shell
Resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obsDiffraction-ID% possible all
2.25-2.330.0151.4194962784198.6
2.33-2.420.0151.9196062758199.8
2.42-2.530.0152.2196152810199.9
2.53-2.670.0152.61868429591100
2.67-2.830.0153.72018927281100
2.83-3.050.0155.2209442852199.9
3.05-3.360.0158.4206462860199.9
3.36-3.840.01512.51876228071100
3.84-4.830.01520.9206262847199.9
4.83-29.4490.01518.7203442869199.3

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SHELXphasing
SHARPphasing
XSCALEDecember 29, 2011data scaling
BUSTER-TNT2.8.0refinement
XDSdata reduction
SHELXDphasing
BUSTER2.8.0refinement
RefinementMethod to determine structure: MAD / Resolution: 2.25→29.449 Å / Cor.coef. Fo:Fc: 0.9241 / Cor.coef. Fo:Fc free: 0.9078 / Occupancy max: 1 / Occupancy min: 0.37 / Cross valid method: THROUGHOUT / σ(F): 0
Details: 1). A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED ...Details: 1). A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 2). PHOSPHATE (PO4) FROM THE CRYSTALLIZATION BUFFER GLYCEROL (GOL), USED AS A CRYOPROTECTANT, AND CHLORIDE (CL) FROM THE PROTEIN BUFFER HAVE BEEN MODELED INTO THE STRUCTURE. 3). ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 4). NCS RESTRAINTS WERE APPLIED USING BUSTER LSSR RESTRAINT REPRESENTATION (-AUTONCS). 5). THE REFINEMENT WAS RESTRAINED AGAINST THE MAD PHASES. 6). UNEXPLAINED DIFFERENCE ELECTRON DENSITY IN THE VICINITY OF LEU 288 ON THE C SUBUNIT COULD NOT BE RELIABLY MODELED.
RfactorNum. reflection% reflectionSelection details
Rfree0.2374 780 5.01 %RANDOM
Rwork0.2133 ---
obs0.2144 15582 --
Displacement parametersBiso max: 101.87 Å2 / Biso mean: 36.462 Å2 / Biso min: 17.14 Å2
Baniso -1Baniso -2Baniso -3
1--2.1845 Å20 Å20 Å2
2---2.1845 Å20 Å2
3---4.3691 Å2
Refinement stepCycle: LAST / Resolution: 2.25→29.449 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1499 0 14 71 1584
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d740SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes49HARMONIC2
X-RAY DIFFRACTIONt_gen_planes220HARMONIC5
X-RAY DIFFRACTIONt_it1555HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion197SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact1831SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d1555HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg2111HARMONIC21.12
X-RAY DIFFRACTIONt_omega_torsion4.55
X-RAY DIFFRACTIONt_other_torsion2.69
LS refinement shellResolution: 2.25→2.4 Å / Total num. of bins used: 8
RfactorNum. reflection% reflection
Rfree0.2588 140 5.12 %
Rwork0.2056 2596 -
all0.2083 2736 -
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.6756-0.482-0.75471.77820.74973.88970.0010.0773-0.0481-0.1248-0.09490.09790.077-0.35240.09390.0102-0.003-0.0013-0.0061-0.0022-0.11325.516215.22071.3069
23.20740.3738-1.90131.7389-0.39974.254-0.0012-0.25450.19330.1096-0.07350.0254-0.11860.4720.0747-0.0327-0.02110.02010.01580.0352-0.130338.440733.2605-3.5318
32.1432-0.2203-1.52762.69350.3355.3017-0.09280.18360.0275-0.1003-0.001-0.0876-0.07230.17980.0938-0.0032-0.01850.0128-0.03950.0203-0.120139.02130.3943-11.5248
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A281 - 340
2X-RAY DIFFRACTION2B281 - 341
3X-RAY DIFFRACTION3C281 - 341

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