Supplemental Experimental Procedures Expression and Purification of Receptor Components

Soluble human GM-CSF was produced in Escherichia coli and purified by anion exchange chromatography and reversed phase HPLC (Hercus et al., 1994). DNA fragments encoding the soluble extracellular domains of GMRα (sGMRα) or βc (sβc) were cloned into the pIBV5-His expression vector (Invitrogen) and transfected into Sf21 insect cells using Cellfectin (Invitrogen). Permanently transfected Sf21 cell lines were selected by incubation with Blasticidin at 50 µg/ml (Invitrogen) and maintained in serum-free ExCell 420 medium (JRH Biosciences) at 27°C. Soluble receptors were purified from conditioned medium by affinity chromatography (McClure et al., 2003). Human βc contains three N-linked glycosylation sites and elimination of the third site at N346 by glutamine substitution has previously been demonstrated to improve the diffraction properties of crystallized sβc (Gustin et al., 2001). We expressed and purified sβc N346Q from Sf21 cells and observed a modest reduction in molecular weight compared to the wild-type sβc but no difference in its ability to form a ternary complex with sGMRα and GM-CSF (results not shown). Crystallization of the Comple