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. 2018 Jan 8;8(1):41.
doi: 10.1038/s41598-017-18570-w.

Towards High Throughput GPCR Crystallography: In Meso Soaking of Adenosine A 2A Receptor Crystals

Free PMC article

Towards High Throughput GPCR Crystallography: In Meso Soaking of Adenosine A 2A Receptor Crystals

Prakash Rucktooa et al. Sci Rep. .
Free PMC article


Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity "carrier" ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).

Conflict of interest statement

The authors are shareholders of Sosei Group Corporation and declare competing financial interests.


Figure 1
Figure 1
A2A-StaR2-b RIL562 Crystal Soaking. (A) Bar chart showing the melting temperature of A2A-StaR2-b RIL562 in its apo form or in the presence of theophylline, ZM241385 or Compound 4e, reflecting the relative stability of each protein preparation. (B) SDS-PAGE of concentrated A2A-StaR2-b RIL562 protein prior to crystallisation. (C) Crystals of the A2A-StaR2-b RIL562-Theophylline complex. (D) A2A-StaR2-b RIL562-Theophylline crystals following a 1 hour soak in 1 mM Compound 4e. (E) A2A-StaR2-b RIL562-Theophylline crystals following a 24 hour soak in 1 mM Compound 4e.
Figure 2
Figure 2
Structure of A2A-StaR2-b RIL562-ligand complexes. (A) Structure of the A2A-StaR2-b RIL562-Theophylline complex (PDB: 5MZJ) shown in cartoon, with helices coloured differently from blue (helix 1) to red (helix 8). Theophylline is shown as sticks within the 1.0 σ contoured 2mFo-dFc electron density maps (blue mesh) carved around the ligand. Interesting orthosteric binding site residues are shown as sticks. 1.0 σ contoured 2mFo-dFc and 3.5 σ contoured mFo-dFc ligand omit electron density maps (blue and green meshes respectively) reflecting the quality of ligand (purple sticks) fitting are shown in the top panel, whereas the lower panel provides interaction details between A2A-StaR2-b RIL562 binding site residues (sticks) with Tozadenant (B), LUAA47070 (C) or Vipadenant (D). In these figures, water molecules are represented as red spheres whereas hydrogen bonding is highlighted by dotted lines. An overlay of structures of ZM241385 in complex with A2A-StaR2-b RIL562 from either a bespoke preparation (PDB: 5UI4) (cyan) or from a soaking experiment (orange), and with A2A b RIL562 (PDB: 4EIY) (white) depicts the high degree of conservation in positioning of orthosteric binding site residues (E). Residues and water molecules involved in ligand binding within a 5 Å radius are represented as sticks and as spheres respectively. Hydrogen bonds are shown as dotted lines and the 1.0 σ contoured 2mFo-dFc and 3.5 σ contoured mFo-dFc ligand omit electron density maps corresponding to ZM241385 from the soaking experiment are represented as blue and green meshes respectively, carved around the ligand. Similarly electron density maps and interactions are shown for A2A-StaR2-b RIL562-Compound 4e generated from bespoke crystallisation (F) or from either 1 hour (G) or 24 hour (H) soaks of A2A-StaR2-b RIL562-Theophylline crystals with Compound 4e.

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