2W3V

MYCOBACTERIUM AVIUM DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND TRIMETHOPRIM

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SSRL BEAMLINE BL9-1
Synchrotron site	SSRL
Beamline	BL9-1
Temperature [K]	100
Detector technology	IMAGE PLATE
Collection date	2000-01-20
Detector	MARRESEARCH
Wavelength(s)	0.785
Spacegroup name	P 41 21 2
Unit cell lengths	70.816, 70.816, 73.613
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	14.830 - 1.890
R-factor	0.182
Rwork	0.180
R-free	0.22400
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1rx1
RMSD bond length	0.014
RMSD bond angle	1.553
Data reduction software	MOSFLM
Data scaling software	SCALA
Phasing software	AMoRE
Refinement software	REFMAC (5.2.0019)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	15.000	1.950
High resolution limit [Å]	1.890	1.890
Rmerge	0.070	0.540
Number of reflections	15319
<I/σ(I)>	11.2	2.7
Completeness [%]	98.2	90.5
Redundancy	4.6	4.1

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	6.5	277	MAVDHFR (15 MG/ML) IN 20 MM HEPES (PH 7.0), 1 MM DTT, 0.1 MM EDTA, 1.5 MM NAN3 WAS MIXED WITH NADPH (3 MM, 15 MIN AT ROOM TEMPERATURE) AND THEN TRIMETHOPRIM (2 MM, 15 MIN ON ICE). CRYSTALLIZATION (HANGING DROP VAPOR PHASE EQUILIBRATION) WAS ACHIEVED BY MIXING WITH AN EQUAL VOLUME OF THE PROTEIN COMPLEX WITH A RESERVOIR SOLUTION CONSISTING OF 70% 2-METHYL-2, 4-PENTANEDIOL (MPD) AND 100 MM HEPES (PH 6.5), AND SUSPENDING THE MIXTURE OVER THE RESERVOIR AT 277 K. SMALL ROD-LIKE CRYSTALS (0.01 X 0.01 X 0.05 MM) GREW WITHIN 2 DAYS. CRYSTALS WERE FLASH-COOLED DIRECTLY FROM THE DROP IN LIQUID N2.