3LQ0

Zymogen structure of crayfish astacin metallopeptidase


X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION, SITTING DROP5.6277For crystallization, reservoir solutions were prepared by a Tecan robot and 200-nL crystallization drops were dispensed on 96x2-well MRC plates (Innovadyne) by a Cartesian (Genomic Solutions) nanodrop robot at the High-Throughput Crystallography Platform of the Barcelona Science Park. Best crystals appeared in a Bruker steady-temperature crystal farm at 4C with protein solution (10 mg/mL in 50mM AMPSO pH9.0) and 20% PEG 8000, 0.1M (NH4)2SO4, 0.01M MgCl2, 0.05M MES pH5.6 as reservoir solution. These conditions were efficiently scaled up to the microliter range with 24-well Cryschem crystallization dishes (Hampton Research). Crystals were cryo-protected with 16% PEG 8000, 20% glycerol, 0.1M (NH4)2SO4, 0.01M MgCl2, 0.05M MES pH5.6. , VAPOR DIFFUSION, SITTING DROP, temperature 277K
Crystal Properties
Matthews coefficientSolvent content
2.3246.94

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 55.91α = 90
b = 63.37β = 90
c = 69.19γ = 90
Symmetry
Space GroupP 21 21 21

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100CCDMAR CCD 165 mm2009-10-06MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONESRF BEAMLINE ID23-20.873ESRFID23-2

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)R Merge I (Observed)R Sym 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
11.4546.7399.80.0440.04427.37.144316442271.44214.6
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)R Merge I (Observed)R-Sym I (Observed)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
11.451.5498.90.4740.4743.86.26285

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Number Reflections (All)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (All)R-Factor (Observed)R-WorkR-FreeR-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUTPDB ENTRY 1AST1.4541.92442844339876899.730.152180.152180.151690.18046RANDOM7.029
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]
-0.69-0.871.56
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg31.382
r_dihedral_angle_4_deg19.977
r_dihedral_angle_3_deg12.504
r_dihedral_angle_1_deg6.213
r_sphericity_free5.332
r_scangle_it3.675
r_scbond_it2.694
r_sphericity_bonded2.085
r_mcangle_it1.791
r_angle_refined_deg1.532
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg31.382
r_dihedral_angle_4_deg19.977
r_dihedral_angle_3_deg12.504
r_dihedral_angle_1_deg6.213
r_sphericity_free5.332
r_scangle_it3.675
r_scbond_it2.694
r_sphericity_bonded2.085
r_mcangle_it1.791
r_angle_refined_deg1.532
r_rigid_bond_restr1.356
r_mcbond_it1.219
r_angle_other_deg0.958
r_mcbond_other0.508
r_chiral_restr0.09
r_bond_refined_d0.016
r_gen_planes_refined0.008
r_bond_other_d0.001
r_gen_planes_other0.001
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms1859
Nucleic Acid Atoms
Solvent Atoms298
Heterogen Atoms24

Software

Software
Software NamePurpose
ProDCdata collection
PHASERphasing
REFMACrefinement
XDSdata reduction
SCALAdata scaling