1TKK

The Structure of a Substrate-Liganded Complex of the L-Ala-D/L-Glu Epimerase from Bacillus subtilis

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 19-ID
Synchrotron site	APS
Beamline	19-ID
Temperature [K]	150
Detector technology	CCD
Collection date	2001-03-01
Detector	CUSTOM-MADE
Wavelength(s)	1.0
Spacegroup name	P 21 21 21
Unit cell lengths	117.400, 134.600, 194.900
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	30.000 - 2.100
R-factor	0.222
Rwork	0.219
R-free	0.28500
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1jpm
RMSD bond length	0.010
RMSD bond angle	1.199
Data reduction software	d*TREK
Data scaling software	d*TREK
Phasing software	MOLREP
Refinement software	REFMAC (5.1.24)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	2.180
High resolution limit [Å]	2.100	2.100
Rmerge	0.159	0.508
Number of reflections	173946
<I/σ(I)>	8.6	2.6
Completeness [%]	96.7	96.7
Redundancy	6.2	6.4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	293	The protein was concentrated to 20 mg/ml and dialyzed against 5 mM HEPES, 2 mM MgCl2, 50 mM NaCl, 0.25 mM TCEP, 1 mM NaN3, pH 7.0, drop frozen as small pellets in liquid nitrogen and stored at -80 C. The frozen concentrated protein was thawed and diluted to 10 mg/mL with 5 mM HEPES, 2 mM MgCl2, 1 mM NaN3, pH 7.5. A stock solution of the peptide substrate L-Ala-L-Glu (Sigma) was neutralized to pH 7.5 with NaOH and brought to a final concentration of 1.6 M. This solution was added to the protein as 1/50th part by volume and the resultant mixture utilized for crystallization experiments. The initial crystals were grown by hanging drop by mixing 5 ?l of protein solution and 5 ?l of a solution containing 9-11% dimethyl-PEG 5000, 50 mM MOPS, 1% MPD, 1 mM NaN3, pH 7.0 and suspending the droplets over 600 ?l precipitant at 20 C, VAPOR DIFFUSION, HANGING DROP, temperature 293K