4X2P

P. putida mandelate racemase in complex with 3-hydroxypyruvate

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 21-ID-G
Synchrotron site	APS
Beamline	21-ID-G
Temperature [K]	100
Detector technology	CCD
Collection date	2014-08-01
Detector	MARMOSAIC 300 mm CCD
Wavelength(s)	0.979
Spacegroup name	I 4 2 2
Unit cell lengths	123.879, 123.879, 105.301
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	40.000 - 1.650
R-factor	0.14471
Rwork	0.143
R-free	0.17677
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.036
RMSD bond angle	3.095
Data reduction software	DENZO
Data scaling software	SCALEPACK
Phasing software	PHASER
Refinement software	REFMAC (5.7.0029)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	1.680
High resolution limit [Å]	1.650	1.650
Rmerge	0.082	0.437
Number of reflections	49040
<I/σ(I)>	13.2	3.2
Completeness [%]	99.6	94
Redundancy	13	8.4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	BATCH MODE	4.5	298	For the crystals grown in the presence of freshly made 3-HP, the batch reservoir consisted of 280 mM KCl, 46% PEP 426, and 140 mM sodium acetate (pH 4.5). The protein solution consisted of 6.0 mg/ml MR purified as described above, 50mM HEPES buffer (pH 7.5), and 3.3 mM MgCl2. 20mM of 3-HP was added to the protein solution before being mixed with the batch reservoir. The protein solution and reservoir solution were gently mixed in a 1:1 ratio. The mixture was pipetted into a lightly greased well to prevent the crystals from adhering to the well bottom and sealed with a glass coverslip lined with mineral oil. The spontaneous crystals grew for 17 hours, resulting in hexagonal prism and octahedral shaped crystals, and were looped directly from the wells to be flash-cooled in liquid nitrogen for storage.