6GKQ

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


X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
TypeSourceAccession CodeDetails
experimental modelPDB 1LCL 

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1IN CELL310Data 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.
Crystal Properties
Matthews coefficientSolvent content
2.7154.55

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 48.941α = 90
b = 48.941β = 90
c = 258.337γ = 120
Symmetry
Space GroupP 65 2 2

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100PIXELDECTRIS EIGER X 16M2015-09-22MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONPETRA III, EMBL c/o DESY BEAMLINE P14 (MX2)0.9763PETRA III, EMBL c/o DESYP14 (MX2)

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)Rrim I (All)CC (Half)Net I Over Average Sigma (I)RedundancyNumber Reflections (All)Number Reflections (Observed)Observed Criterion Sigma (F)Observed Criterion Sigma (I)B (Isotropic) From Wilson Plot
12.225099.90.310.997.8511.86997223.36
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)Rrim I (All)CC (Half)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
12.222.3599.10.1950.481.2912.2

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (Observed)R-WorkR-FreeR-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUT1LCL2.343.0689848961000.1910.1870.232RANDOM29.85
Temperature Factor Modeling
Anisotropic B[1][1]Anisotropic B[1][2]Anisotropic B[1][3]Anisotropic B[2][2]Anisotropic B[2][3]Anisotropic B[3][3]
-5.737-5.73711.4741
RMS Deviations
KeyRefinement Restraint Deviation
t_other_torsion17.52
t_omega_torsion4.27
t_angle_deg1.13
t_bond_d0.009
t_dihedral_angle_d
t_incorr_chiral_ct
t_pseud_angle
t_trig_c_planes
t_gen_planes
t_it
RMS Deviations
KeyRefinement Restraint Deviation
t_other_torsion17.52
t_omega_torsion4.27
t_angle_deg1.13
t_bond_d0.009
t_dihedral_angle_d
t_incorr_chiral_ct
t_pseud_angle
t_trig_c_planes
t_gen_planes
t_it
t_nbd
t_improper_torsion
t_chiral_improper_torsion
t_sum_occupancies
t_utility_distance
t_utility_angle
t_utility_torsion
t_ideal_dist_contact
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms1110
Nucleic Acid Atoms
Solvent Atoms113
Heterogen Atoms6

Software

Software
Software NamePurpose
BUSTERrefinement
XDSdata reduction
XDSdata scaling
PHASERphasing