5C7J

CRYSTAL STRUCTURE OF NEDD4 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	2013-08-15
Detector	ADSC QUANTUM 315
Wavelength(s)	0.97918
Spacegroup name	P 32
Unit cell lengths	139.867, 139.867, 59.386
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	50.010 - 3.000
R-factor	0.2283
Rwork	0.227
R-free	0.27659
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2znv
RMSD bond length	0.007
RMSD bond angle	1.116
Data scaling software	HKL-3000
Phasing software	MOLREP
Refinement software	REFMAC (5.8.0123)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	50.000	50.000	3.050
High resolution limit [Å]	3.000	8.130	3.000
Rmerge	0.071	0.043	0.979
Rmeas	0.074	0.047
Rpim	0.028	0.018	0.370
Total number of observations	198521
Number of reflections	26035
<I/σ(I)>	8.7
Completeness [%]	100.0	99.8	100
Redundancy	7.6	6.8	7.8
CC(1/2)		0.997	0.754

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	293	The NEDD4 and Ubiquitin Variant was mixed at molar ratio ~1:2, and then concentrated to 15mg/ml. The protein sample was mixed with Trypsin at a 1:1000 (W/W) Trypsin:protein ratio before setting up crystallization. Crystal was initially obtained from Molecular Dimentions Proplex screen condition E04. Crystal used for structure refinement was grown in 20% PEG8000, 10% Glycerol, 0.1M HEPES pH 7.0 in hanging drop setup, using 1.5uL protein, 1.5uL well solution over 0.5 mL reservoir buffer at 20 C. Crystals grow to mountable size in 4 days. Harvested crystal was flash-frozen in liquid nitrogen. 20% Glycerol was used as the cryo-protectant