3NPU

Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7.5	292	0.2M NaF, 0.1M Bis Tris Propane, 20% PEG 3350, pH 7.5, VAPOR DIFFUSION, SITTING DROP, temperature 292K

Crystal Properties
Matthews coefficient	Solvent content
2.28	46.03

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 62.222	α = 90
b = 53.295	β = 110.19
c = 81.846	γ = 90

Symmetry
Space Group	P 1 21 1

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	IMAGE PLATE	RIGAKU RAXIS IV++	mirros	2008-06-22	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	ROTATING ANODE	RIGAKU RU300	1.5418

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)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	2.25	50	97.8	0.081	0.077	22.28	6.5		23599	51193		30.25

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)	Redundancy	Number Unique Reflections (All)
1	2.25	2.33	83.8		0.217	0.195	4.8	4.9	2034

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	1JCJ	2.25	50	22380	1207	97.28	0.19198	0.18927	0.24116	RANDOM	30.257

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]
0.81		-1.22	0.14		-1.8

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	31.272
r_dihedral_angle_4_deg	17.007
r_dihedral_angle_3_deg	15.891
r_dihedral_angle_1_deg	7.94
r_scangle_it	4.105
r_scbond_it	2.672
r_angle_refined_deg	1.733
r_mcangle_it	1.515
r_mcbond_it	0.944
r_nbtor_refined	0.293

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	31.272
r_dihedral_angle_4_deg	17.007
r_dihedral_angle_3_deg	15.891
r_dihedral_angle_1_deg	7.94
r_scangle_it	4.105
r_scbond_it	2.672
r_angle_refined_deg	1.733
r_mcangle_it	1.515
r_mcbond_it	0.944
r_nbtor_refined	0.293
r_symmetry_vdw_refined	0.218
r_nbd_refined	0.199
r_symmetry_hbond_refined	0.198
r_xyhbond_nbd_refined	0.131
r_chiral_restr	0.122
r_bond_refined_d	0.02
r_gen_planes_refined	0.007

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3651
Nucleic Acid Atoms
Solvent Atoms	41
Heterogen Atoms

Software

Software
Software Name	Purpose
HKL-2000	data collection
PHASER	phasing
REFMAC	refinement
HKL-2000	data reduction
SCALEPACK	data scaling

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.