6GZH

Crystal Structure of Human CDK9/cyclinT1 with A86

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE MASSIF-1
Synchrotron site	ESRF
Beamline	MASSIF-1
Temperature [K]	100.15
Detector technology	PIXEL
Collection date	2017-03-02
Detector	DECTRIS PILATUS3 2M
Wavelength(s)	0.966
Spacegroup name	H 3
Unit cell lengths	171.599, 171.599, 97.790
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	30.000 - 3.170
R-factor	0.17497
Rwork	0.171
R-free	0.25677
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3blh
RMSD bond length	0.007
RMSD bond angle	1.187
Data scaling software	SCALA
Phasing software	REFMAC
Refinement software	REFMAC (5.8.0158)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	3.340
High resolution limit [Å]	3.170	3.170
Rmeas	0.156	0.702
Number of reflections	16344	2244
<I/σ(I)>	3.6	1.1
Completeness [%]	89.7	83.7
Redundancy	1.5	1.4

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.3	277.15	CDK9/Cyclin T1 at a concentration of 4.5 mg/ml (20 mM Tris / HCl, 250 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.3) was pre-incubated with 0.6 mM (5.1-fold molar excess) of A-86 (150 mM in DMSO) and 4 mM TCEP for 1 h. 0.1 ul of the protein solution was then mixed 0.1 ul of reservoir solution (0.01 M Ca-Chloride, 0.1 M MES/NaOH, pH 6.50, 1.2 M Na-Acetate) and equilibrated at 4 C over 0.06 ml of reservoir solution. Crystals were obtained using microseeding. Well diffracting crystals appeared within 4 days and grew to full size over 8 days