1KMV

HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND (Z)-6-(2-[2,5-DIMETHOXYPHENYL]ETHEN-1-YL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE (SRI-9662), A LIPOPHILIC ANTIFOLATE

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	1998-06-18
Detector	BRANDEIS - B4
Spacegroup name	P 21 21 21
Unit cell lengths	41.300, 54.990, 82.680
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	20.000 - 1.050
Rwork	0.130
R-free	0.18100 *
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	UNPUBLISHED HUMAN DHFR STRUCTURE.
RMSD bond length	0.017
RMSD bond angle	2.270 *
Data reduction software	MOSFLM
Data scaling software	CCP4 ((SCALA))
Phasing software	AMoRE
Refinement software	SHELXL-97

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	20.000	1.090
High resolution limit [Å]	1.050	1.050
Rmerge	0.053	0.231
Total number of observations	312507 *
Number of reflections	86349
<I/σ(I)>	9.6	2.8
Completeness [%]	97.5	83.8
Redundancy	3.6	2.1

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7 *	4 *	THE TERNARY COMPLEX OF DHFR WITH NADPH AND SRI-9662 WAS FORMED BY MIXING HUMAN DHFR (20 MG/ML IN 25 MM KPO4 (PH 7.0), 0.1 MM EDTA, AND 3 MM NAN3) WITH 60 MM NADPH, FOLLOWED 15 MINUTES LATER BY 60 MM OF INHIBITOR IN DMSO (FINAL CONCENTRATIONS OF 2 MM NADPH AND 2 MM INHIBITOR). THIS COMPLEX SOLUTION WAS MIXED WITH AN EQUAL VOLUME OF PRECIPITANT CONTAINING 24-33% PEG 4000, 0.2 M LI2SO4, 0.1 M TRIS CL (PH 7.9-8.4), 5% GLYCEROL, AND EQUILIBRATED WITH THE PRECIPITANT BY HANGING DROP VAPOR DIFFUSION AT 277 K. THE CRYSTAL GREW IN ABOUT 3 WEEKS. THE CRYSTAL WAS FLASH-COOLED DIRECTLY IN LIQUID NITROGEN AFTER HARVESTING INTO MOTHER LIQUOR CONTAINING 10% GLYCEROL., pH 8.00, VAPOR DIFFUSION, HANGING DROP

Crystallization Reagents in Literatures

ID	crystal ID	solution	reagent name	concentration (unit)	details
1	1	drop	hDHFR	20 (mg/ml)
10	1	reservoir	glycerol	5 (%(v/v))	for hDHFR/NADPH/SRI-9662 ternary complex
2	1	drop		25 (mM)	pH7.0
3	1	drop	EDTA	0.1 (mM)
4	1	drop		3 (mM)
5	1	drop	NADPH	2 (mM)
6	1	drop	inhibitor	2 (mM)
7	1	reservoir	PEG4000	24-33 (%(w/v))
8	1	reservoir		0.2 (M)
9	1	reservoir	Tris-HCl	0.1 (M)	pH7.9-8.4