6TP8

Substrate protein interactions in the limbus region of the catalytic site of Candida antarctica Lipase B

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	1TCA

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, HANGING DROP	4.8	293	Crystallization trials were performed at 293 K using the hanging-drop method using a Qiagen EasyXtal 15-well plate. A 15 mg.mL CALB solution in 20mM Na(CH3COO) pH = 4.8 was incubated with a solution of paraoxon-ethyl (SIGMA catalog n. N12816) 30mM in water for one day. 1 uL of CALB solution was diluted with 1 uL of the precipitant solution, made of 200mM Na(CH3COO) pH = 4.8, 20% (w.v) PEG4000, and 15% (v.v) glyceryl tributyrate (SIGMA catalog n. T8626). The drop was equilibrated by vapor diffusion against 500 uL of the precipitant solution. Protein crystals of the CALB complex appeared within two weeks and grew to a size of 0.1 x 0.1 x 0.05 mm3 in five weeks.

Crystal Properties
Matthews coefficient	Solvent content
2.45	49.7

Crystal Data

Unit Cell
Length ( ? )	Angle ( ? )
a = 89.426	�� = 90
b = 156.654	�� = 90
c = 138.084	�� = 90

Symmetry
Space Group	C 2 2 21

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	PIXEL	DECTRIS PILATUS 6M		2013-09-22	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	EMBL/DESY, HAMBURG BEAMLINE X13	0.824	EMBL/DESY, HAMBURG	X13

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	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	1.55	67.69	99.18	0.121	0.14	0.05	0.997	8.4	7.6		138806		1	18.9

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.55	1.605	98.05		1.46	1.7	0.877	0.55	1	7.2

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Cut-off Sigma (F)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	FREE R-VALUE	1TCA	1.55	67.69	1.34	138777	7001	99.18	0.1644	0.163	0.1921	21.85

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
Key	Refinement Restraint Deviation
f_dihedral_angle_d	5.6247
f_angle_d	0.9043
f_chiral_restr	0.0522
f_plane_restr	0.0064
f_bond_d	0.0057

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	6972
Nucleic Acid Atoms
Solvent Atoms	1461
Heterogen Atoms	150

Software

Software
Software Name	Purpose
PHENIX	refinement
XDS	data reduction
Aimless	data scaling
MOLREP	phasing

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 R01GM157729.