9QV4

1-Phosphofructokinase mutant K95T/G110S from E. coli with bound fructose-1,6-bisphosphate and ADP

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	PETRA III, EMBL c/o DESY BEAMLINE P14 (MX2)
Synchrotron site	PETRA III, EMBL c/o DESY
Beamline	P14 (MX2)
Temperature [K]	100
Detector technology	PIXEL
Collection date	2020-12-11
Detector	DECTRIS EIGER X 16M
Wavelength(s)	0.97625
Spacegroup name	I 2 2 2
Unit cell lengths	63.120, 98.210, 140.100
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	19.740 - 1.550
R-factor	0.1603
Rwork	0.160
R-free	0.17420
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.007
RMSD bond angle	1.012
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	PHENIX
Refinement software	PHENIX ((1.21.2_5419: ???))

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	19.740	19.740	1.590
High resolution limit [Å]	1.550	6.930	1.550
Rmerge	0.055	0.026	0.831
Rmeas	0.058	0.027	0.872
Number of reflections	63211	762	4550
<I/σ(I)>	25.55
Completeness [%]	99.6
Redundancy	13.4
CC(1/2)	1.000	1.000	0.833

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6.5	289	The FruK variant K95T/G110S (3.5 mg/mL) in 20 mM MOPS/KOH buffer (pH 7.4) containing 75 mM KCl and 1 M L-proline was mixed in a 1:1 ratio with condition containing 26 % (w/v) pentaerythritol ethoxylate, 75 mM MES (pH 6.5), 150 mM ammonium sulfate, 8 mM fructose-6-phosphate, 4 mM ADP, and 10 mM magnesium chloride. The final drop volume was 0.8 microliters. Before plunge freezing in liquid nitrogen, the crystals were soaked with addintional 20 mM fructose-6-phosphate, 2 mM ADP, 5 mM magnesium chloride for about 1 minute, before glycerol was added as a cryo-protectant to a final concentration of 30% (v/v).