9G1H

Fragment screening of FosAKP, room-temperature structure in complex with fragment F2X-entry H01

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	PETRA III, DESY BEAMLINE P09 HiPhaX
Synchrotron site	PETRA III, DESY
Beamline	P09 HiPhaX
Temperature [K]	296.15
Detector technology	PIXEL
Collection date	2023-07-15
Detector	DECTRIS PILATUS 6M-F
Wavelength(s)	0.7749
Spacegroup name	P 21 21 2
Unit cell lengths	71.480, 91.590, 45.250
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	56.350 - 1.400
R-factor	0.1493
Rwork	0.148
R-free	0.16780
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.005
RMSD bond angle	0.883
Data reduction software	CrystFEL (0.10.1)
Data scaling software	CrystFEL (0.10.1)
Phasing software	PHENIX (1.20-4459_9999)
Refinement software	PHENIX (1.20-4459_9999)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	91.600	1.450
High resolution limit [Å]	1.400	1.400
Number of reflections	59357	5839
<I/σ(I)>	6.1605	1.25
Completeness [%]	100.0	100
Redundancy	101.16	29.5
CC(1/2)	0.967	0.512

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	291	25 mg/mL FosAKP in 10 mM Hepes, pH 7.5, 75 mM NaCl was supplemented with 6 mM MnCl2 and mixed with an equal volume of 16% (w/v) PEG3350, 0.25 M MgCl2, 0.2 M KBr, 0.1 M BisTris, pH 5.5 and 1/10 volume of seed stock in 26% (w/v) PEG3350, 0.25 M MgCl2, 0.2 M KBr, 0.1 M BisTris, pH 5.5. Approximately of this solution 14 uL were added per window of the fixed-target chip. The sample holder was then inserted for into a 3D-printed crystal growth chamber with 3 mL of precipitant solution in the bottom for vapor-diffusion crystallization and incubated at 20C. For fragment application sample holders were removed from the crystal growth chamber and excess precipitant was removed by blotting through the micropores of the membranes, before 10 uL of fragment solution at a concentration of 25 mM in 5% DMSO were pipetted to the crystals in the individual compartments. Sample holders were then placed back into the growths vessel and incubated for 24h. Before data collection sample holder was removed from the crystal growth chamber and excess precipitant was removed by blotting through the micropores of the membranes, before 10 uL of crystallization solution with 5% DMSO were pipetted to the crystals in the individual compartments. Sample holders were then placed back into the growths vessel and incubated for 24h. Before data collection blotting was repeated for removal of excess liquid in order to minimize background scattering. Sample holders were then equipped with a protective cover to prevent them from drying-out and stored in a humid atmosphere. Compound addition and liquid removal were conducted in a glove box with >95% rel. humidity.