T of approximately 20 kDa were pooled. The pooled fractions were concentrated using Amicon ultrafiltration cell. The concentrated protein was passed through Sephadex G100 column (10062 cm) using 50 mM Tris-HCl pH 8.0. Two peaks were obtained when the fractions were read at 280 nm wavelength. The purity of the eluted fractions was checked using SDS-PAGE which indicated that the second peak in the gel filtration profile corresponded to the molecular weight of 20 kDa of PGRP-S. The high molecular weight first peak also showed a band at about 20 kDa on the SDS-PAGE indicating a polymeric state of CPGRP-S.Materials and Methods Ethics StatementThe peripheral blood was taken from the PF-00299804 healthy human volunteers with the approval of the Institute Ethics Committee atTable 1. Data collection and refinement statistics for the structure of the ternary complex of CPGRP-S with LPS and SA.PGRP-S+LPS+SA PDB ID Space group Unit cell dimensions ?a (A) ?b (A) ?c (A) Number of molecules in the asymmetric unit ?Vm (A3/Da) Solvent Content ( ) ?Resolution range (A) Number of unique reflections#4 GF9 I89.6 101.9 162.3 4 2.32 47.0 50.0?.8 37206 7.9 (28.9) 33.9 (4.3) 98.8 (99.5) 22.9 26.6 5348 256 48 20 6 0.02 1.9 18.0 11.4 12.7 12.Rsym ( )Binding StudiesFreshly purified sample of CPGRP-S was immobilized on a CM5 carboxyldextran chip using carbodiimide chemistry to a level of 10000 response units (RU) as described previously (9, 10) using a BIAcore-T200 (BIAcore). In one experiment three different concentrations (200 nM, 150 nM, and 100 nM) of analytes, SA and LPS were passed over the immobilized CPGRP-S at a flow rate of 10 ml/min with an injection time 24272870 of 420 seconds. The regeneration of bound analytes was done using 10 mM NaOH for 100 seconds at a flow rate of 30 ml/min. The association (kon) and dissociation constants (koff) for the binding of analytes to CPGRP-S were calculated and values of equilibrium constants were obtained using mass action relation KD = koff/kon with BIA evaluation software provided by the manufacturer. In another experiment only binding analysis of both the analytes was done by passing both the analytes one after the other with injection time of 420 seconds and flow rate of 10 ml/min. However, the regeneration procedure was carried only once at the end of experiment. The resulted sensogram was recorded which showed that both analytes bound to CPGRP-S in the presence of each other.I/s(I) Overall completeness of data ( ) *Rcryst ( ) Rfree ( ) Protein atoms Water oxygen atoms Atoms of LPS Atoms of SA Atoms of glycerol ?R.m.s.d in bond lengths (A) R.m.s.d in bond Crenolanib site angles (u) R.m.s.d in torsion angles (u) ?Mean B-factor for main chain atoms (A2) ?Mean B-factor for side chain atoms (A2) ?Mean B-factor for all atoms(A2) Ramachandran’s w, y map Residues in the most favoured regions ( ) Residues in the additionally allowed regions ( )90.2 9.Induction of TNF-a and IFN-c by LPS and SAThe peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll Hypaque gradient centrifugation and suspended in complete RPMI-1640 with 10 fetal calf serum (FCS) at optimum culture conditions of 5 CO2, at 37uC for 6 hrs. Cells were stimulated with medium alone and with 10 mg/ ml LPS and SA without and with 10 mg/ml CPGRP-S. TheThe values in parentheses correspond to the values in the highest resolution shell. # Rsym = ghklgi | Ii(hkl) ?,I(hkl). |/ghkl gi Ii(hkl). *Rcryst = ghkl | Fo(hkl) 2 Fc(hkl) |/ghkl | Fo(hkl) | where Fo and Fc are o.T of approximately 20 kDa were pooled. The pooled fractions were concentrated using Amicon ultrafiltration cell. The concentrated protein was passed through Sephadex G100 column (10062 cm) using 50 mM Tris-HCl pH 8.0. Two peaks were obtained when the fractions were read at 280 nm wavelength. The purity of the eluted fractions was checked using SDS-PAGE which indicated that the second peak in the gel filtration profile corresponded to the molecular weight of 20 kDa of PGRP-S. The high molecular weight first peak also showed a band at about 20 kDa on the SDS-PAGE indicating a polymeric state of CPGRP-S.Materials and Methods Ethics StatementThe peripheral blood was taken from the healthy human volunteers with the approval of the Institute Ethics Committee atTable 1. Data collection and refinement statistics for the structure of the ternary complex of CPGRP-S with LPS and SA.PGRP-S+LPS+SA PDB ID Space group Unit cell dimensions ?a (A) ?b (A) ?c (A) Number of molecules in the asymmetric unit ?Vm (A3/Da) Solvent Content ( ) ?Resolution range (A) Number of unique reflections#4 GF9 I89.6 101.9 162.3 4 2.32 47.0 50.0?.8 37206 7.9 (28.9) 33.9 (4.3) 98.8 (99.5) 22.9 26.6 5348 256 48 20 6 0.02 1.9 18.0 11.4 12.7 12.Rsym ( )Binding StudiesFreshly purified sample of CPGRP-S was immobilized on a CM5 carboxyldextran chip using carbodiimide chemistry to a level of 10000 response units (RU) as described previously (9, 10) using a BIAcore-T200 (BIAcore). In one experiment three different concentrations (200 nM, 150 nM, and 100 nM) of analytes, SA and LPS were passed over the immobilized CPGRP-S at a flow rate of 10 ml/min with an injection time 24272870 of 420 seconds. The regeneration of bound analytes was done using 10 mM NaOH for 100 seconds at a flow rate of 30 ml/min. The association (kon) and dissociation constants (koff) for the binding of analytes to CPGRP-S were calculated and values of equilibrium constants were obtained using mass action relation KD = koff/kon with BIA evaluation software provided by the manufacturer. In another experiment only binding analysis of both the analytes was done by passing both the analytes one after the other with injection time of 420 seconds and flow rate of 10 ml/min. However, the regeneration procedure was carried only once at the end of experiment. The resulted sensogram was recorded which showed that both analytes bound to CPGRP-S in the presence of each other.I/s(I) Overall completeness of data ( ) *Rcryst ( ) Rfree ( ) Protein atoms Water oxygen atoms Atoms of LPS Atoms of SA Atoms of glycerol ?R.m.s.d in bond lengths (A) R.m.s.d in bond angles (u) R.m.s.d in torsion angles (u) ?Mean B-factor for main chain atoms (A2) ?Mean B-factor for side chain atoms (A2) ?Mean B-factor for all atoms(A2) Ramachandran’s w, y map Residues in the most favoured regions ( ) Residues in the additionally allowed regions ( )90.2 9.Induction of TNF-a and IFN-c by LPS and SAThe peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll Hypaque gradient centrifugation and suspended in complete RPMI-1640 with 10 fetal calf serum (FCS) at optimum culture conditions of 5 CO2, at 37uC for 6 hrs. Cells were stimulated with medium alone and with 10 mg/ ml LPS and SA without and with 10 mg/ml CPGRP-S. TheThe values in parentheses correspond to the values in the highest resolution shell. # Rsym = ghklgi | Ii(hkl) ?,I(hkl). |/ghkl gi Ii(hkl). *Rcryst = ghkl | Fo(hkl) 2 Fc(hkl) |/ghkl | Fo(hkl) | where Fo and Fc are o.
