4YXA

Complex of SpaO(SPOA1,2 SeMet) and OrgB(APAR)::T4lysozyme fusion protein

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	NSLS BEAMLINE X29A
Synchrotron site	NSLS
Beamline	X29A
Temperature [K]	100
Detector technology	CCD
Collection date	2014-03-12
Detector	ADSC QUANTUM 315r
Wavelength(s)	0.979
Spacegroup name	P 1 21 1
Unit cell lengths	62.880, 88.500, 63.320
Unit cell angles	90.00, 116.07, 90.00

Refinement procedure

Resolution	45.804 - 2.350
R-factor	0.2027
Rwork	0.198
R-free	0.26180
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	4yx7
RMSD bond length	0.011
RMSD bond angle	1.464
Data scaling software	Aimless (0.2.7)
Phasing software	PHENIX
Refinement software	PHENIX

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	47.610	47.610	2.430
High resolution limit [Å]	2.350	9.100	2.350
Rmerge	0.088	0.056	0.617
Rpim	0.037	0.023	0.256
Total number of observations	169948	2593	16809
Number of reflections	25759
<I/σ(I)>	12.8	29.5	2.6
Completeness [%]	99.0	86.5	99.6
Redundancy	6.6	6.4	6.6
CC(1/2)	0.997	0.997	0.816

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		293	SpaO(145-213, SeMet) + SpaO (232-297, SeMet) + OrgB(1-30)::T4 lysozyme (native) was concentrated to 18mg/mL, supplemented with 50mM maltose, and crystallized with 25% PEG3350, 200mM ammonium formate, 100mM sodium acetate pH=5.0. Microseeding was employed to enhance crystal uniformity and diffraction. Briefly, crystals to be seeded were harvested in precipitant solution and vortexed in a microfuge tube with a small stir bar for ~60 seconds. The slurry of microseeds was serially dilluted (5-10-fold steps) in precipitant solution and 5 selected microseed-precipitant mixtures were mixed with fresh protein as in a normal hanging drop experiment. Crystals were cryoprotected in 25% PEG3350, 10% ethylene glycol, 200mM ammonium formate, 100mM sodium acetate pH=5.0, 50mM maltose.