2VT1

Crystal structure of the cytoplasmic domain of Spa40, the specificity switch for the Shigella flexneri Type III Secretion System

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE ID23-1
Synchrotron site	ESRF
Beamline	ID23-1
Temperature [K]	100
Detector technology	CCD
Collection date	2008-04-13
Detector	ADSC CCD
Spacegroup name	P 1
Unit cell lengths	25.035, 30.753, 32.099
Unit cell angles	102.52, 110.97, 94.30

Refinement procedure

Resolution	29.590 - 2.000
R-factor	0.18
Rwork	0.178
R-free	0.22700
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3BZL WITH SIDECHAINS MUTATED TO SER
RMSD bond length	0.010
RMSD bond angle	1.105
Data reduction software	MOSFLM
Data scaling software	SCALA
Phasing software	PHASER
Refinement software	REFMAC (5.4.0077)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.050
High resolution limit [Å]	2.000	2.000
Rmerge	0.110	0.400
Number of reflections	5433
<I/σ(I)>	3.8	1.7
Completeness [%]	93.2	93.3
Redundancy	2.6	2.7

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7	293	SITTING DROPS CONTAINING 200 NL PROTEIN (3.3 MG/ML IN 20 MM TRIS PH 8.0, 500 MM NACL) AND 200 NL RESERVOIR SOLUTION (0.1M HEPES PH7.0, 0.2M NH4CL AND 20% PEG W/V 6000) WERE EQUILIBRATED AGAINST 100 UL RESERVOIRS AT 20 C. CRYSTALS WERE CRYOPROTECTED IN RESERVOIR SOLUTION SUPPLEMENTED WITH 25% (V/V) GLYCEROL. THE ASYMMETRIC UNIT VOLUME IS NOT SUFFICIENT TO ACCOMMODATE THE ENTIRE SPA40 CONSTRUCT. WE ASSUME THAT THE PROTEIN HAS UNDERGONE SOME PROTEOLYSIS ADDITIONAL TO THE SELF-CLEAVAGE BETWEEN RESIDUES 257 AND 258, REMOVING EITHER THE DISORDERED N-TERMINUS OR THE C-TERMINAL HIS TAG. THE SOLVENT CONTENT QUOTED IS FOR RESIDUE 237 TO THE END OF THE CONSTRUCT.