6GKQ

X-ray structure determined from ex vivo Charcot-Leyden crystal

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	2015-09-22
Detector	DECTRIS EIGER X 16M
Wavelength(s)	0.9763
Spacegroup name	P 65 2 2
Unit cell lengths	48.941, 48.941, 258.337
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	43.060 - 2.300
R-factor	0.191
Rwork	0.187
R-free	0.23200
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1lcl
RMSD bond length	0.009
RMSD bond angle	1.130
Data reduction software	XDS
Data scaling software	XDS
Phasing software	PHASER
Refinement software	BUSTER (2.10.2)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	2.350
High resolution limit [Å]	2.220	2.220
Rmeas	0.310	0.195
Number of reflections	9972	1533
<I/σ(I)>	7.85	1.29
Completeness [%]	99.9	99.1
Redundancy	11.86	12.2
CC(1/2)	0.990	0.480

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	IN CELL		310	Data was collected from ex vivo Charcot-Leyden crystals. Sticky allergic mucin type mucus was obtained from CRSwNP patients undergoing endoscopic sinus surgery. One g of the allergic mucin was cut thoroughly in 10 ml RPMI-1640 (Sigma-Aldrich) containing antibiotics (50 IU/mL penicillin and 50 mg/mL streptomycin; One g of the allergic mucin was cut thoroughly in 10 ml RPMI-1640 (Sigma-Aldrich) containing antibiotics (50 IU/mL penicillin and 50 mg/mL streptomycin; Thermo Fisher Scientific), 0.1 percent BSA (Sigma-Aldrich) and 1 mg/ml Collagen type 2 (Worthington), and further homogenized using a GentleMACS Dissociator (Myltenyi Biotec) and subsequently incubed at 37 degrees for 45 minutes under continuous rotation. After centrifugation the pellet was dissolved in 3 ml PBS containing antibiotics, to which 6 ml Ficoll-Paque (GE Healthcare) was added. After centrifugation at 250g and removal of the supernatant and most of the Ficoll layer, the pellet was dissolved 1:10 in PBS with antibiotics. This precipitation process was repeated 5 more times. The final pellet containing the crystals was resuspended in PBS with antibiotics.