Table 1

Crystallographic statistics for unliganded NPR-C and the NPR-C/CNP complex. The human NPR-C receptor extracellular domain (430 amino acids) was secreted from D. melanogaster cells, and the COOH-terminal hexa-histidine tag was removed with carboxypeptidase-A (40). Crystals of unliganded NPR-C were grown from 1.5 M sodium/potassium phosphate or 1.5 M potassium phosphate (pH 7.5) and cryo-cooled in the presence of 20% ethylene glycol. For the complex, NPR-C was incubated with an excess of human CNP and then purified by gel filtration chromatography. Crystals of the complex were grown from 0.8 M sodium citrate (pH 7.5) and were cryo-cooled in 20% glycerol. Data from a single crystal for both unliganded and complex were collected at SSRL BL9-2 and 9-1, respectively, and processed with MOSFLM and SCALA (41). The higher resolution NPR-C/CNP complex was solved first by molecular replacement (MR) with the coordinates of the unliganded ECD of rat NPR-A (Protein Data Bank accession code, 1DP4) (22). Only MOLREP (41) identified correct solutions with a polyalanine model. The two receptor monomers were refined in the early stages with NCS restraints, which were then released to allow for differences on surface loops. The hormone CNP was traced in a single unique conformation in two equally occupied twofold symmetric orientations, reflecting its location along the receptor dimer twofold. The peptide was refined in two orientations with equal occupancy (0.5) to satisfactory stereochemical criteria, with all but one (Met17) residue falling within favored or allowed regions of the Ramachandran plot as determined by PROCHECK (41). The unliganded NPR-C structure was solved by MR with the separate NH2- and COOH-terminal domains of the refined NPR-C complex. Both liganded and unliganded structures were refined with a maximum-likelihood target function, rigid-body refinement, cycles of simulated annealing with torsion angle molecular dynamics, and iterations between positional and B-factor minimization. SIGMAA-weighted 2Fo-Fc and omit Fo-Fc maps (CNS) were manually rebuilt in the program O, and stereochemical analysis was performed with PROCHECK (41). All data were used without truncation, except for 5% data randomly selected for cross validation. In the unliganded structure, two asparagine-linked glycosylation sites contained electron density for a total of 11 glycan residues (6 for Asn248, 5 for Asn349). In the NPR-C/CNP complex, we modeled a total of five N-linked glycan moieties on two N-linked sites in each monomer (Asn248 and Asn349). In both structures, one chloride ion was clearly defined in each monomer.

Unliganded NPR-CNPR-C/CNP complex
Data
Space groupP6122P212121
Unit cell (Å) (a, b, c )217.19, 217.19, 130.9856.80, 136.46, 137.59
Matthews coefficient (Å3/dalton)7.42.2
SourceSSRL, BL9-2SSRL, BL9-1
Resolution (Å) (highest resolution shell)50.0–2.9 (3.0–2.9)50.0–2.0 (2.07–2.0)
Measured reflections217,841225,890
Unique reflections43,72270,832
Completeness (%)98.1 (98.4)97.7 (96.6)
I/σ(I)7.4 (2.0)6.7 (1.6)
Rmerge* (%)8.1 (43.5)7.4 (44.8)
Refinement statistics
Resolution range (Å)50.0–2.9 (3.0–2.9)50.0–2.0 (2.07–2.0)
R cryst 0.243 (0.367)0.227 (0.330)
R free 0.256 (0.389)0.248 (0.343)
Average B factors (Å2)
NPR-C66.742.4
CNP72.9
Oligosaccharides82.582.7
Waters46.0
Chlorides34.930.6
Root mean square deviation from ideality
Bond lengths (Å)0.0090.009
Bond angles (°)1.41.5
BondedB factors (Å2) (main chain, side chain)2.2, 3.21.9, 2.5
Ramachandran plot (%)
(Favored, allowed, generous, disallowed)85.9, 13.3, 0.8, 088.3, 11.2, 0.5, 0
  • * Rmerge = Σhkl|I − 〈I〉∣/ΣhklI, where I is the intensity of unique reflection hkl, and 〈I〉 is the average over symmetry-related observation of unique reflectionhkl.

  • Rcryst = Σ∣FobsFcalcFobs, where Fobs andFcalc are the observed and the calculated structure factors, respectively.

  • Rfree is Rwith 5% of reflections sequestered before refinement.