2W3W

MYCOBACTERIUM AVIUM DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND A LIPOPHILIC ANTIFOLATE SELECTIVE FOR M. AVIUM DHFR, 6-((2,5- DIETHOXYPHENYL)AMINOMETHYL)-2,4-DIAMINO-5-METHYLPYRIDO(2,3-D) PYRIMIDINE (SRI-8686)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	NSLS BEAMLINE X25
Synchrotron site	NSLS
Beamline	X25
Temperature [K]	100
Detector technology	CCD
Collection date	2000-04-13
Detector	BRANDEIS UNIVERSITY BRANDEIS B4
Spacegroup name	P 21 21 21
Unit cell lengths	36.729, 53.965, 81.191
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	14.980 - 1.600
R-factor	0.175
Rwork	0.173
R-free	0.20700
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	UNPUBLISHED M. AVIUM DHFR-NADPH- TRIMETHOPRIM COMPLEX SINCE DEPOSITED AS PDB ENTRY 2W3V.
RMSD bond length	0.014
RMSD bond angle	1.664
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	1.640
High resolution limit [Å]	1.600	1.600
Rmerge	0.080	0.460
Number of reflections	18299
<I/σ(I)>	10.8	1.7
Completeness [%]	83.5	37.9
Redundancy	4.5	1.2

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	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 SRI-8686 (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 55-70% 2-METHYL-2, 4-PENTANEDIOL (MPD) AND 100 MM HEPES (PH 6.5-7.5), 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.