9MFN

Crystal Structure of ADI-64596 (human Fab, with substituted IgG1-CH1 (HC-L145Q, K147E, and S181E) and substituted kappa constant domain (LC-T129R, T178R, and T180Q))

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	DIAMOND BEAMLINE I04
Synchrotron site	Diamond
Beamline	I04
Temperature [K]	100
Detector technology	PIXEL
Collection date	2021-05-12
Detector	DECTRIS EIGER2 XE 16M
Wavelength(s)	0.9795
Spacegroup name	P 31
Unit cell lengths	65.594, 65.594, 96.795
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	48.990 - 2.350
R-factor	0.2089
Rwork	0.208
R-free	0.22140
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.002
RMSD bond angle	0.551
Data reduction software	XDS
Data scaling software	Aimless
Phasing software	PHASER
Refinement software	PHENIX ((1.18.2_3874: ???))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	96.790	2.430
High resolution limit [Å]	2.350	2.350
Number of reflections	208056	19723
<I/σ(I)>	7.9	1
Completeness [%]	100.0	100
Redundancy	10.7	10.1
CC(1/2)	0.997	0.537

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	8.5	293	ADI-64596 (human Fab) was received at a concentration of 11.35 mg/mL in a buffer containing 2 mM Tris-HCl pH 8.0 and 150 mM NaCl. The PACT, BCS, and JCSG+ screens (all from Molecular Dimensions Ltd.) were initially set up using a mosquito crystallization robot (STP Labtech). Since crystals obtained from these initial screens only gave rise to low-resolution X-ray diffraction, crystal seed solutions were prepared and applied in the setup of the BCS, PACT, and Additive Screens (Hampton Research). Sitting drops of 160 nL protein and 160 nL precipitant solution were left to equilibrate against a 40 uL reservoir at 20 degrees C. After a few days, plate- and needle-like crystals appeared in several conditions. The precipitant solution giving rise to the best-diffracting crystal contained 75 mM Tris pH 8.5, 25 mM Bis-Tris-propane pH 8.5, 22.5% (v/v) PEG Smear Low, 5% (w/v) PEG3350, and 50 mM NaBr