2C2T

Human Dihydrofolate Reductase Complexed With NADPH and 2,4-Diamino-5-((7,8-dicarbaundecaboran-7-yl)methyl)-6-methylpyrimidine, a novel boron containing, nonclassical Antifolate

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	1999-03-03
Detector	MARRESEARCH
Spacegroup name	C 2 2 21
Unit cell lengths	87.628, 94.354, 96.032
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	20.000 - 1.500
R-factor	0.16
Rwork	0.158
R-free	0.21300
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	UNPUBLISHED HUMAN DHFR STRUCTURE
RMSD bond length	0.012
RMSD bond angle	1.451
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]	35.000	1.540
High resolution limit [Å]	1.500	1.500
Rmerge	0.060	0.450
Number of reflections	62594
<I/σ(I)>	8.4	1.7
Completeness [%]	98.4	90.1
Redundancy	2.9	2.1

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	8	277	THE TERNARY COMPLEX OF DHFR WITH NADPH AND 39B 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. NOTE THAT THE INHIBITOR IS ACTUALLY A RACEMIC MIXTURE OF 39B AND ITS ENANTIOMER, HERE CALLED 39E. IT IS THE RACEMATE THAT IS BOUND IN THE CRYSTAL, REPRESENTED IN THE COORDINATES AS A 1:1 STATISTICALLY-DISORDERED MIXTURE OF 39B AND 39E.