5VLO

The structure of human CamKII with bound inhibitor

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ALS BEAMLINE 5.0.2
Synchrotron site	ALS
Beamline	5.0.2
Temperature [K]	100
Detector technology	PIXEL
Collection date	2016-02-25
Detector	DECTRIS PILATUS3 6M
Wavelength(s)	1.0
Spacegroup name	P 1 21 1
Unit cell lengths	54.473, 67.546, 85.450
Unit cell angles	90.00, 94.11, 90.00

Refinement procedure

Resolution	47.380 - 2.050
R-factor	0.1837
Rwork	0.183
R-free	0.22610
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.003
RMSD bond angle	0.518
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	EPMR
Refinement software	PHENIX (1.10.1_2155)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	2.170
High resolution limit [Å]	2.050	2.050
Number of reflections	38959	5733
<I/σ(I)>	12.7	1.63
Completeness [%]	98.5	91
Redundancy	13	12.7
CC(1/2)	0.998	0.742

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP		293	CamKII S3-K301, in 20mM imidazole pH 8.5, 0.3M sodium chloride, 5mM TCEP, was concentrated to 36 mg/ml and flash frozen in liquid nitrogen for long term storage at -80 C in 10 uL aliquots. The protein was thawed and diluted down to 12 mg/mL in the same buffer just prior to crystallization experiments. Sitting drop vapor diffusion droplets were assembled with 250 nL of 12 mg/mL CamKII, 0.6 mM inhibitor and 250 nL of reservoir solution 24% peg 3350, 0.2 M ammonium tartrate, 0.1 M arginine. Flat crystal plates (typically 0.03 mm x 0.2 mm x 0.4 mm in size) grew in 4-7 days at 20 C. A crystal seed suspension was prepared with ten crushed crystals combined into 100 uL of reservoir solution and stored at -80 C. A thirty fold seed dilution was prepared in the same solution for addition to protein droplets in a 1 to 1 volume ratio to enhance crystallization of difficult to crystallize inhibitors.