6NY0

Crystal structure of trimethoprim-resistant type II dihydrofolate reductase in complex with a bisbenzimidazole inhibitor

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	CLSI BEAMLINE 08ID-1
Synchrotron site	CLSI
Beamline	08ID-1
Temperature [K]	93
Detector technology	CCD
Collection date	2011-08-12
Detector	RAYONIX MX300HE
Wavelength(s)	0.9795
Spacegroup name	I 41 2 2
Unit cell lengths	67.492, 67.492, 51.791
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	41.090 - 1.400
R-factor	0.142
Rwork	0.141
R-free	0.15770
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2rh2
RMSD bond length	0.018
RMSD bond angle	2.076
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	REFMAC
Refinement software	REFMAC (5.8.0238)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	50.000	50.000	1.370
High resolution limit [Å]	1.350	3.660	1.350
Rmerge	0.102	0.058
Total number of observations	271880
Number of reflections	13545	757	671
<I/σ(I)>	8.8
Completeness [%]	100.0	99.6	100
Redundancy	20.1	19.1	14.6

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8	277	The protein was concentrated to 20 mg/mL in 100 mM Tris pH 8.0. Immediately before crystallization, chymotrypsin was added to the sample in a ratio of 1:100 chymotrypsin:protein, and the protein was diluted to 15 mg/mL using MPD, resulting in a final MPD concentration of 25%. Reservoirs were prepared using 750 uL of 100 mM sodium phosphate pH 7.6 and 60% MPD in a Greiner 24-well hanging-drop crystallization plate. On a siliconized glass cover slip (Hampton Research), 1.5 uL of protein were combined with 2.5 uL of reservoir solution and suspended over the well. The plate was incubated at 277 K and crystals were obtained in a few days.