5O4C

From macrocrystals to microcrystals: a strategy for membrane protein serial crystallography

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	FREE ELECTRON LASER
Source details	SLAC LCLS BEAMLINE CXI
Synchrotron site	SLAC LCLS
Beamline	CXI
Temperature [K]	293
Detector technology	PIXEL
Collection date	2015-04-15
Detector	CS-PAD CXI-1
Wavelength(s)	1.89
Spacegroup name	P 43 21 2
Unit cell lengths	226.400, 226.400, 113.700
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	36.900 - 2.800
R-factor	0.16665
Rwork	0.165
R-free	0.19612
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	5nj4
RMSD bond length	0.015
RMSD bond angle	2.052
Data reduction software	CrystFEL (0.6.2)
Data scaling software	CrystFEL (0.6.2)
Phasing software	PHASER
Refinement software	REFMAC (5.8.0158)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	36.900	2.890
High resolution limit [Å]	2.800	2.800
Number of reflections	69325
<I/σ(I)>	10.4	0.88
Completeness [%]	100.0	100
Redundancy	1342	361
CC(1/2)	0.997	0.408

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6.8	277	Crystals grown from one round of seeding. Macrocrystal growth: 25 ul sitting drop. 10 ul 10 mg/ml protein, 15 ul precipitate solution Precipitate solution: 3.6 M Ammonium sulphate, 20 mM sodium phosphate buffer pH 6.8. 6 % w:v heptane-1-2-3-triol. 1 ml 2 M Ammonium sulphate reservoir Growth at 277 K for 4 days. Macrocrystals crushed into seed stock Micro-crystals 17.5 ul sitting drop. 10 ul 8.5 mg/ml protein, 7.5 ul precipitate solution 1 ml 2 M Ammonium sulphate solution