2VT1

Crystal structure of the cytoplasmic domain of Spa40, the specificity switch for the Shigella flexneri Type III Secretion System


X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION, SITTING DROP7293SITTING DROPS CONTAINING 200 NL PROTEIN (3.3 MG/ML IN 20 MM TRIS PH 8.0, 500 MM NACL) AND 200 NL RESERVOIR SOLUTION (0.1M HEPES PH7.0, 0.2M NH4CL AND 20% PEG W/V 6000) WERE EQUILIBRATED AGAINST 100 UL RESERVOIRS AT 20 C. CRYSTALS WERE CRYOPROTECTED IN RESERVOIR SOLUTION SUPPLEMENTED WITH 25% (V/V) GLYCEROL. THE ASYMMETRIC UNIT VOLUME IS NOT SUFFICIENT TO ACCOMMODATE THE ENTIRE SPA40 CONSTRUCT. WE ASSUME THAT THE PROTEIN HAS UNDERGONE SOME PROTEOLYSIS ADDITIONAL TO THE SELF-CLEAVAGE BETWEEN RESIDUES 257 AND 258, REMOVING EITHER THE DISORDERED N-TERMINUS OR THE C-TERMINAL HIS TAG. THE SOLVENT CONTENT QUOTED IS FOR RESIDUE 237 TO THE END OF THE CONSTRUCT.
Crystal Properties
Matthews coefficientSolvent content
1.6726.55

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 25.035α = 102.52
b = 30.753β = 110.97
c = 32.099γ = 94.3
Symmetry
Space GroupP 1

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100CCDADSC CCDMIRRORS2008-04-13MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONESRF BEAMLINE ID23-1ESRFID23-1

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)R Merge I (Observed)Net I Over Average Sigma (I)RedundancyNumber Reflections (All)Number Reflections (Observed)Observed Criterion Sigma (F)Observed Criterion Sigma (I)B (Isotropic) From Wilson Plot
123093.20.113.82.65433-321.6
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)R Merge I (Observed)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
122.0593.30.41.72.7

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (Observed)R-WorkR-FreeR-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUTPDB ENTRY 3BZL WITH SIDECHAINS MUTATED TO SER229.59518125293.20.180.1780.227RANDOM18.59
Temperature Factor Modeling
Anisotropic B[1][1]Anisotropic B[1][2]Anisotropic B[1][3]Anisotropic B[2][2]Anisotropic B[2][3]Anisotropic B[3][3]
-1.29-0.4-1.061.110.92-0.24
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg34.309
r_dihedral_angle_3_deg15.846
r_dihedral_angle_4_deg13.76
r_scangle_it5.385
r_dihedral_angle_1_deg4.8
r_scbond_it3.273
r_mcangle_it2.211
r_mcbond_it1.21
r_angle_refined_deg1.105
r_angle_other_deg0.752
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg34.309
r_dihedral_angle_3_deg15.846
r_dihedral_angle_4_deg13.76
r_scangle_it5.385
r_dihedral_angle_1_deg4.8
r_scbond_it3.273
r_mcangle_it2.211
r_mcbond_it1.21
r_angle_refined_deg1.105
r_angle_other_deg0.752
r_symmetry_vdw_other0.235
r_symmetry_hbond_refined0.101
r_xyhbond_nbd_refined0.095
r_symmetry_vdw_refined0.092
r_chiral_restr0.06
r_nbd_other0.048
r_nbd_refined0.031
r_bond_refined_d0.01
r_gen_planes_refined0.004
r_bond_other_d0.001
r_gen_planes_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_other
r_mcangle_other
r_scbond_other
r_scangle_other
r_long_range_B_refined
r_long_range_B_other
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms806
Nucleic Acid Atoms
Solvent Atoms29
Heterogen Atoms

Software

Software
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing