5C7M

CRYSTAL STRUCTURE OF E3 LIGASE ITCH WITH A UB VARIANT

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 19-ID
Synchrotron site	APS
Beamline	19-ID
Temperature [K]	100
Detector technology	CCD
Collection date	2013-03-07
Detector	ADSC QUANTUM 315
Wavelength(s)	0.97899
Spacegroup name	P 3 2 1
Unit cell lengths	121.115, 121.115, 85.547
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	49.430 - 3.030
R-factor	0.2554
Rwork	0.254
R-free	0.29690
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3tug
RMSD bond length	0.008
RMSD bond angle	0.830
Data reduction software	HKL-3000
Data scaling software	HKL-2000
Phasing software	BALBES
Refinement software	BUSTER (2.10.2)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	50.000	50.000	3.070
High resolution limit [Å]	3.020	8.190	3.020
Rmerge	0.094	0.067	0.908
Number of reflections	14296
<I/σ(I)>	12.5
Completeness [%]	99.9	98.5	100
Redundancy	18.1	15.8	18.5

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7.5	293	The ITCH and ubiquitin variant ubv.it.02 were mixed at molarity ratio 1:2, and then concentrated to 17mg/ml. The protein sample was mixed with 1mg/mL chymotrypsin at a 1:1000 (W/W) chymotrypsin:protein ratio right before set up crystallization. Crystal was initially obtained from SGC-I screen condition A05. Crystal used for structure refinement was grown in 1.6M NH4SO4, 0.2M NaAc, 0.1M HEPES pH 7.5, 5% Ethylene Glycol in hanging drop setup, using 1.2uL protein, 1.2uL well solution over 0.5 mL reservoir buffer at 20 C. Crystals grow to mountable size for ~1 weeks. Harvested crystal was flash-frozen in liquid nitrogen. A well solution containing 20% glycerol was used as the cryo-protectant