8AF5

Room temperature SSX structure of GH11 xylanase from Nectria haematococca (10000 frames)


X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
TypeSourceAccession CodeDetails
experimental modelPDB 6Y0H 

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1BATCH MODE5.5293For crystallization, the original conditions were modified to obtain microcrystals. Initial crystals obtained from hanging drops under the precipitant condition: 1 M ammonium sulfate, 100 mM sodium citrate pH 5.5 were crushed under a stereomicroscope, using a crystal crusher tool (Hampton research). The reservoir solution was pipetted to the drop and the seed stock was collected by washing the drop with reservoir solution. The seed stock was transferred to a seed bead tube (Molecular Dimensions Ltd., UK), vortexed three times for 30 s each, with an interval of 30 s between each vortex, to get the final seed stock. Protein solution 15 mg/mL, precipitant solution, and seed stock were mixed with a ratio of 1:1:0.5. The mixture was vortexed for 30 s in ten-minute intervals. After 30 min, the microcrystals were centrifuged at 200 rpm and the supernatant was replaced with a precipitant solution. Applying the same protocol, microcrystals were obtained under two precipitant conditions i.e. Precipitant 1: 1M (NH4)2SO4, 100 mM sodium citrate pH 5.5, and Precipitant 2: 200 mM (NH4)2SO4, 100 mM sodium citrate pH 5.5 and 20% PEG 6000. Microcrystals obtained under both precipitant conditions were tested for diffraction data collection.
Crystal Properties
Matthews coefficientSolvent content
2.0439.61

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 80.55α = 90
b = 38.85β = 91
c = 53.57γ = 90
Symmetry
Space GroupC 1 2 1

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray295PIXELDECTRIS PILATUS 6M2019-05-14MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONPETRA III, DESY BEAMLINE P111.0332PETRA III, DESYP11

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)CC (Half)R Split (All)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
11.6317.51000.960.2263.671342089620.59
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)CC (Half)R Split (All)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
11.631.660.1891.680.66

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Cut-off Sigma (F)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (Observed)R-WorkR-FreeMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTFREE R-VALUE6Y0H1.6317.161.3420856161899.820.16620.16250.210423.57
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]
RMS Deviations
KeyRefinement Restraint Deviation
f_dihedral_angle_d6.3865
f_angle_d1.0682
f_chiral_restr0.0612
f_bond_d0.0099
f_plane_restr0.0068
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms1459
Nucleic Acid Atoms
Solvent Atoms233
Heterogen Atoms

Software

Software
Software NamePurpose
PHENIXrefinement
CrystFELdata reduction
CrystFELdata scaling
PHENIXphasing