1T4M

STRUCTURE OF A THERMOSTABLE DOUBLE MUTANT OF BACILLUS SUBTILIS LIPASE OBTAINED THROUGH DIRECTED EVOLUTION

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP	9.5		PEG 3350, ethanolamine, n-octyl-beta-D-glucoside, sodium sulfate., pH 9.5, VAPOR DIFFUSION, HANGING DROP, temperature 100K

Crystal Properties
Matthews coefficient	Solvent content
3	58.9

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 76.015	α = 90
b = 76.015	β = 90
c = 103.093	γ = 120

Symmetry
Space Group	H 3

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	MARRESEARCH	osmic	2003-04-03	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 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	94	0.044	19	2.1	14102	14102			27.9

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R-Sym I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
	2	2.07	63.2		0.241	2.4	1.9	793

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (All)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (All)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	PDB ENTRY 1I6W	2	24.19	14102	14102	712	94.2	0.207	0.207	0.206	0.257	RANDOM	32.2

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]
3.8	3.77		3.8		-7.6

RMS Deviations
Key	Refinement Restraint Deviation
c_dihedral_angle_d	24
c_angle_deg	1.2
c_improper_angle_d	0.7
c_bond_d	0.006

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	1358
Nucleic Acid Atoms
Solvent Atoms	183
Heterogen Atoms	1

Software

Software
Software Name	Purpose
DENZO	data reduction
SCALEPACK	data scaling
MOLREP	phasing
CNS	refinement

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.