Receptor expression is conducted in parallel in four cell types (HEK, CHO, Sf9, and S2) and two constructs in order to identify the most suitable format for large scale purification. The objective at this step is to reach an expression level compatible with the crystallisation screening, meaning mg/l yields. This step is charged only if an expression yield in excess of a million receptors per cell is achieved. The number of receptors is estimated by western blot and by radio ligand binding when available. The functionality of the target of interest will also be assessed by additional means where suitable (e.g. gene reporter, patch clamp, calcium release…)
Receptor purification starts with the screening of solubilisation conditions in detergents compatible with crystallisation conditions (with respect to size and homogeneity). The receptor monodispersity in detergent is assessed by analytical gel filtration. The receptor conformation integrity is also controlled using ligand binding assay (when available). When ligand binding properties are lost by the receptor in detergent, lipid vesicle reconstitution screening is performed to retrieve it. Large scale purification is conducted using the best solubilisation conditions to obtain mg quantities of receptor in monodisperse form. As an additional step, directed proteolysis can lead to the improvement of the monodispersity of the receptor.
The availability of a strong affinity binder facilitates the structure resolution of membrane receptors in three ways: 1) it increases the polar surface of the receptor, thus enabling the formation of new crystal bonds while mitigating the problems of flexible loops; 2) it can stabilize the receptor in a single conformation, thus increasing the homogeneity of the sample; and 3) it can increase the solubility of the receptor, thus facilitating its purification.
Finally, the selection of binders may lead to the discovery of functional molecules that can be useful as pharmacological tools for target validation or even for therapeutic applications.
The selected binders are characterized to determine their affinity, potential functionality, induced improvement of receptor stability, solubility and monodispersity. This step is charged for only if binders with nanomolar range affinity are discovered.
Crystallization screenings are performed using both vapour diffusion and lipid cubic phase methods in the presence of the best selected binders or combinations of binders. The best crystal hits are optimized so that high resolution data can be collected at a synchrotron radiation facility. This step is charged for only when a high resolution structure can be obtained.