7C8I

Ambient temperature structure of Bifidobacgterium longum phosphoketolase with thiamine diphosphate and phosphoenol pyuruvate

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	FREE ELECTRON LASER
Source details	SACLA BEAMLINE BL3
Synchrotron site	SACLA
Beamline	BL3
Temperature [K]	293
Detector technology	CCD
Collection date	2016-10-13
Detector	MPCCD
Wavelength(s)	1.7714
Spacegroup name	P 1 21 1
Unit cell lengths	145.300, 184.940, 163.130
Unit cell angles	90.00, 99.18, 90.00

Refinement procedure

Resolution	48.100 - 2.500
R-factor	0.169
Rwork	0.166
R-free	0.22570
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3ai7
RMSD bond length	0.009
RMSD bond angle	1.630
Data reduction software	CrystFEL (0.6.1)
Data scaling software	CrystFEL (0.6.1)
Phasing software	MOLREP (11.0.05)
Refinement software	REFMAC (5.8.0238)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	48.100	2.590
High resolution limit [Å]	2.500	2.500
Number of reflections	292114	29155
<I/σ(I)>	4.16	1.06
Completeness [%]	100.0	100
Redundancy	486.3	343.7
CC(1/2)	0.960	0.411

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	BATCH MODE		298	Equal amount of 15 mg ml-1 PKT solution (4 mM DTT, 10 mg ml-1 ThDpp) and precipitant solution (50 mM malonate, 12% tacsimate (pH5), 17% PEG3350, 140 mM PEP) were mixed in order to prepare seed crystals. After 16 h, precipitated crystals were homogenized and centrifuged to use supernatant as seed solution. For the purpose of forming micro-sized crystals of PKT, 1176 micro l of protein-precipitant solution mixture as the same components of seed crystals preparation was mixed with 120 micro l of the seed solution in a 1.5 ml tube. After gentle rotation at 25 degree C for 2 h, the supernatant was exchanged to the same precipitant solution. The same buffer exchange was iteratively conducted after 10 h and 16 h.