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- PDB-6gkq: X-ray structure determined from ex vivo Charcot-Leyden crystal -

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Basic information

Entry
Database: PDB / ID: 6gkq
TitleX-ray structure determined from ex vivo Charcot-Leyden crystal
ComponentsGalectin-10
KeywordsSUGAR BINDING PROTEIN / Galectin-10 / ex vivo crystal
Function / homology
Function and homology information


regulation of activated T cell proliferation / regulation of T cell cytokine production / T cell apoptotic process / regulation of T cell anergy / cysteine-type endopeptidase activity involved in apoptotic process / carbohydrate binding / collagen-containing extracellular matrix / identical protein binding / cytosol
Similarity search - Function
Galectin-like / Galactoside-binding lectin / Galectin / Galectin, carbohydrate recognition domain / Galactoside-binding lectin / Galactoside-binding lectin (galectin) domain profile. / Jelly Rolls - #200 / Concanavalin A-like lectin/glucanase domain superfamily / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsVerstraete, K. / Verschueren, K. / Savvides, S.N.
CitationJournal: Science / Year: 2019
Title: Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.
Authors: Emma K Persson / Kenneth Verstraete / Ines Heyndrickx / Elien Gevaert / Helena Aegerter / Jean-Michel Percier / Kim Deswarte / Koen H G Verschueren / Ann Dansercoer / Delphine Gras / Pascal ...Authors: Emma K Persson / Kenneth Verstraete / Ines Heyndrickx / Elien Gevaert / Helena Aegerter / Jean-Michel Percier / Kim Deswarte / Koen H G Verschueren / Ann Dansercoer / Delphine Gras / Pascal Chanez / Claus Bachert / Amanda Gonçalves / Hanne Van Gorp / Hans De Haard / Christophe Blanchetot / Michael Saunders / Hamida Hammad / Savvas N Savvides / Bart N Lambrecht /
Abstract: Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such ...Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.
History
DepositionMay 21, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 5, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_source / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Galectin-10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,5922
Polymers16,5001
Non-polymers921
Water2,018112
1
A: Galectin-10
hetero molecules

A: Galectin-10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,1844
Polymers33,0002
Non-polymers1842
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_555-y,-x,-z+1/61
Buried area2250 Å2
ΔGint-9 kcal/mol
Surface area11750 Å2
MethodPISA
Unit cell
Length a, b, c (Å)48.941, 48.941, 258.337
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522
Components on special symmetry positions
IDModelComponents
11A-314-

HOH

21A-342-

HOH

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Components

#1: Protein Galectin-10 / Gal-10 / Charcot-Leyden crystal protein / CLC / Eosinophil lysophospholipase / Lysolecithin acylhydrolase


Mass: 16499.887 Da / Num. of mol.: 1 / Mutation: Ala28Val (Natural variant) / Source method: isolated from a natural source
Details: This X-ray data was collected from an ex vivo Charcot-Leyden crystal.
Source: (natural) Homo sapiens (human) / References: UniProt: Q05315
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 112 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.71 Å3/Da / Density % sol: 54.55 %
Crystal growTemperature: 310 K / Method: in cell
Details: 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 ...Details: 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.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Sep 22, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.22→50 Å / Num. obs: 9972 / % possible obs: 99.9 % / Redundancy: 11.86 % / Biso Wilson estimate: 23.36 Å2 / CC1/2: 0.99 / Rrim(I) all: 0.31 / Net I/σ(I): 7.85
Reflection shellResolution: 2.22→2.35 Å / Redundancy: 12.2 % / Mean I/σ(I) obs: 1.29 / Num. unique obs: 1533 / CC1/2: 0.48 / Rrim(I) all: 0.195 / % possible all: 99.1

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Processing

Software
NameVersionClassification
BUSTER2.10.2refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1LCL

1lcl


Resolution: 2.3→43.06 Å / Cor.coef. Fo:Fc: 0.919 / Cor.coef. Fo:Fc free: 0.878 / SU R Cruickshank DPI: 0.237 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.304 / SU Rfree Blow DPI: 0.215 / SU Rfree Cruickshank DPI: 0.197
RfactorNum. reflection% reflectionSelection details
Rfree0.232 896 9.97 %RANDOM
Rwork0.187 ---
obs0.191 8984 100 %-
Displacement parametersBiso mean: 29.85 Å2
Baniso -1Baniso -2Baniso -3
1--5.737 Å20 Å20 Å2
2---5.737 Å20 Å2
3---11.4741 Å2
Refine analyzeLuzzati coordinate error obs: 0.28 Å
Refinement stepCycle: 1 / Resolution: 2.3→43.06 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1110 0 6 113 1229
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0091151HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.131558HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d402SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes31HARMONIC2
X-RAY DIFFRACTIONt_gen_planes163HARMONIC5
X-RAY DIFFRACTIONt_it1151HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion4.27
X-RAY DIFFRACTIONt_other_torsion17.52
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion143SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact1302SEMIHARMONIC4
LS refinement shellResolution: 2.3→2.57 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.237 257 10.45 %
Rwork0.205 2203 -
all0.208 2460 -
obs--100 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.8822-1.49120.3019-0.48080.87521.40220.0428-0.05140.080.0027-0.03330.0251-0.01480.0034-0.00950.00560.0361-0.02560.0176-0.0159-0.0175-35.706621.443111.353
21.76220.1909-0.58090.69140.93180.03980.0941-0.11050.103-0.06610-0.0359-0.0728-0.007-0.09410.0170.02150.0067-0.0626-0.02050.0121-24.294222.961311.9216
31.66120.60270.47463.21430.20151.1009-0.0513-0.0533-0.06650.07660.06140.07510.0244-0.0997-0.0101-0.0123-0.0141-0.0079-0.0114-0.00650.0103-26.443111.35698.9139
41.3574-0.1056-0.3421.31651.13312.58190.1069-0.0482-0.0578-0.05860.0557-0.16750.12660.2108-0.1625-0.0233-0.0096-0.0099-0.0269-0.00160.0109-16.618514.079512.5139
5-0.87120.48110.59812.1657-0.59890.1247-0.0730.04860.09170.01090.0501-0.0409-0.09110.01460.02290.01950.01020.0179-0.0212-0.0130.0024-22.66625.63636.4543
61.46050.51050.6081.77290.23941.63540.03380.17370.0262-0.16610.0473-0.1289-0.12620.0197-0.0811-0.0131-0.00480.0027-0.0521-0.0131-0.0081-21.092716.53263.12
72.42810.424-1.7449-0.3197-0.39470.0775-0.01630.01770.1320.02180.02270.0379-0.0560.0235-0.00640.08610.00860.0112-0.04120.0003-0.0137-30.983725.43117.3967
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|2 - A|11 }
2X-RAY DIFFRACTION2{ A|12 - A|34 }
3X-RAY DIFFRACTION3{ A|35 - A|57 }
4X-RAY DIFFRACTION4{ A|58 - A|87 }
5X-RAY DIFFRACTION5{ A|88 - A|95 }
6X-RAY DIFFRACTION6{ A|96 - A|129 }
7X-RAY DIFFRACTION7{ A|130 - A|138 }

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