8QMS

Succinic semialdehyde dehydrogenase from E. coli with Q262R substitution and bound NAD+

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	2023-03-23
Detector	DECTRIS EIGER2 X CdTe 16M
Wavelength(s)	0.9763
Spacegroup name	P 21 21 21
Unit cell lengths	91.300, 115.690, 179.610
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	29.460 - 1.900
R-factor	0.1818
Rwork	0.181
R-free	0.21330
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.004
RMSD bond angle	0.728
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	PHENIX ((1.20.1_4487))
Refinement software	PHENIX ((1.20.1_4487: ???))

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	29.460	29.460	1.950
High resolution limit [Å]	1.900	8.490	1.900
Rmerge	0.133	0.044	0.998
Rmeas	0.138	0.046	1.036
Number of reflections	149467	1809	10908
<I/σ(I)>	13.34
Completeness [%]	99.5
Redundancy	13.8
CC(1/2)	0.999	0.999	0.920

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	8.25	295	35.0 mg/mL of SAD_Q262R in 20 mM HEPES-KOH, 50 mM KCl, pH 7.5 was supplemented with 5 mM NAD+ and 1 mM TCEP. The enzyme was then mixed in a 1:1 ratio with 285 mM Bis-Tris-Propane, 8.25 pH , 17 % (w/v) PEG4000. The final drop size was 4 uL. The drop was complemented with PEG200 to a final concentration of 16 % (v/v) before flash freezing the crystals in liquid nitrogen.