1C1W

RECRUITING ZINC TO MEDIATE POTENT, SPECIFIC INHIBITION OF SERINE PROTEASES

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Temperature [K]	298
Detector technology	IMAGE PLATE
Collection date	1998-02-18
Detector	RIGAKU RAXIS IV++
Spacegroup name	C 1 2 1
Unit cell lengths	71.220, 71.880, 72.650
Unit cell angles	90.00, 100.69, 90.00

Refinement procedure

Resolution	7.500 - 1.900
R-factor	0.203
Rwork	0.203
R-free	0.24200
Structure solution method	DIFFERENCE FOURIER PLUS REFINEMENT
RMSD bond length	0.019
RMSD bond angle	4.100
Data reduction software	bioteX ((MSC))
Data scaling software	bioteX
Phasing software	X-PLOR
Refinement software	X-PLOR (3.1)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	35.900	1.980
High resolution limit [Å]	1.900	1.900
Rmerge	0.116	0.246
Number of reflections	25689
<I/σ(I)>	5.5	2.1
Completeness [%]	65.0	36.7
Redundancy	1.6

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	unknown *	9		THROMBIN WAS PURCHASED FROM HAEMATOLOGIC TECHNOLOGIES, INC. AND ACETYL-HIRUDIN FROM BACHEM. THROMBIN WAS PREPARED AS DESCRIBED (SKRZPCZAK-JANKUN ET AL., 1991) .THROMBIN (1.0 MG/ML IN 50 MM HEPES, 50 % GLYCEROL, PH 7.0) WAS INCUBATED WITH 1.0 MM ACETYL-HIRUDIN, 1.0 MM BABIM, 1.0 MM ZN+2 FOR 1 HR AT 4 DEG C. GLYCEROL WAS REMOVED AND THE COMPLEX CONCENTRATED WITH A CENTRICON 10 (AMICON) TO 8.6 MG/ML AS DETERMINED BY THE BIORAD PROTEIN ASSAY KIT USING BOVINE SERUM ALBUMIN. CRYSTALS OF THROMBIN-ACETYL-HIRUDIN-BABIM-ZN+2 WERE GROWN IN HANGING DROPS BY VAPOR DIFFUSION AFTER STREAK SEEDING. THE DROPS WERE MADE FROM 5 MICROLITERS OF COMPLEX AND 5 MICROLITERS OF RESERVOIR SOLUTION (0.10 M TRIS, 0.50 M NACL, 22 % (BY VOLUME) PEG 4K, PH 7.00). A CO-CRYSTAL WAS SOAKED IN 30 % PEG 4K,0.50 M NACL, 0.10 M TRIS, 0.4 MM ZN+2, PH 9.00, SATURATED IN KETO- BABIM AND CONTAINING 2 % DMSO.