4GL7

Structure of human placental aromatase complexed with designed inhibitor HDDG046 (compound 5)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	CHESS BEAMLINE A1
Synchrotron site	CHESS
Beamline	A1
Temperature [K]	100
Detector technology	CCD
Collection date	2011-11-12
Detector	ADSC QUANTUM 210
Wavelength(s)	0.978
Spacegroup name	P 32 2 1
Unit cell lengths	141.445, 141.445, 118.851
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	50.000 - 3.900
R-factor	0.21615
Rwork	0.214
R-free	0.25407
Structure solution method	FOURIER SYNTHESIS
RMSD bond length	0.012
RMSD bond angle	1.474
Data reduction software	HKL-2000
Data scaling software	HKL-2000
Phasing software	CCP4
Refinement software	REFMAC (5.5.0109)

Data quality characteristics

	Overall
Low resolution limit [Å]	122.490
High resolution limit [Å]	3.900
Rmerge	0.114
Number of reflections	12126
<I/σ(I)>	16.7
Completeness [%]	99.5
Redundancy	4.6

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.4	277	The enzyme-inhibitor complexes were prepared by the addition from 20mM stock solutions of compound 5 in PEG550 to 18 M (~1mg/ml) of aromatase, to give a final inhibitor concentration of 300 microM. The mixture was incubated overnight at 4 C in 100mM potassium phosphate buffer pH 7.4 containing 20% glycerol, 20mM dithiothreitol, 0.5 microM ASD and 1mM BDM. The complex was then concentrated to 25-30mg/ml using ultrafiltration. Protein was setup for crystallization using protein to cocktail ratios 2:1 and 3:1. The protein was mixed with reservoir cocktails of 24-30% polyethylene glycol 4000 in 50mM NaCl, 50mM Tris, pH 8.5 and vapor diffused in sealed 24-well sitting drop plates against corresponding reservoir solution, VAPOR DIFFUSION, SITTING DROP, temperature 277K