GemSpot: A Pipeline for Robust Modeling of Ligands into CryoEM Maps

Abstract

<jats:title>Abstract</jats:title><jats:p>Producing an accurate atomic model of biomolecule-ligand interactions from maps generated by cryo-electron microscopy often presents challenges inherent to the methodology and the dynamic nature of ligand binding. Here we have developed GemSpot, a pipeline of computational chemistry methods that take into account EM map potentials, quantum mechanics energy calculations, and water molecule site prediction to generate candidate poses and provide a measure of the degree of confidence. The pipeline is validated through several published cryoEM structures of complexes in different resolution ranges and various types of ligands. In all cases, at least one identified pose produced both excellent interactions with the target and agreement with the map. GemSpot will be valuable for the robust identification of ligand poses and drug discovery efforts through cryoEM.</jats:p>

Macromolecular refinement of X-ray and cryo-electron microscopy structures with Phenix / OPLS3e for improved structure and ligand quality

“…To determine the impact of the OPLS3e/VSGB force field in cryo-EM structure refinement, 15 protein-ligand complexes were retrieved from the PDB and EMDB containing pharmaceutically active compounds, a subset of cases we used in our recent GemSpot pipeline (Robertson et al, 2020). Structures were manually prepared using the Protein Preparation Wizard in Maestro.…”

“…After validating our implementation in reciprocal space refinement using X-ray diffraction data, we applied our protocol on 15 deposited cryo-EM structures and maps, previously used in our GemSpot pipeline (Robertson et al, 2020). Since cryo-EM refinement lacks a robust crossvalidation metric, we performed a grid scan over the map weight parameters for our cases, using both the Phenix standard restraints model and the OPLS3e force field.…”

“…The Phenix/OPLS3e implementation has already seen applications in cryo-EM enabled SBDD of macrocycles in ribosomes (Qi et al, 2019) and determining high-confidence ligand binding poses in ambiguous cryo-EM structures in the GemSpot pipeline (Robertson et al, 2020). We foresee our approach as an accessible and user-friendly implementation of a high quality force field to improve structure and ligand quality especially when using lower resolution data provided either by crystallography and cryo-EM, and as an additional measure for reducing overfitting in cryo-EM structure refinement and cryo-EM ligand fitting.…”

Structures of metabotropic GABABreceptor

“…It was apparent from the map that the lipid had only two carbon chains, immediately excluding cardiolipin as it has four hydrocarbon tails. A comparison of the map and the binding site residues led to the decision to model PE (b) into the pocket using the GemSpot pipeline 21 , which produced good cross-correlation with favorable interactions. To further confirm our selection, analysis of overlays of phosphatidylcholine (c) and phosphatidylinositol (d) over the docked model revealed phosphatidylcholine is unlikely given that the interactions with the cation appear to be primarily salt bridges, rather than the cation-π interactions that more commonly coordinate choline in proteins 62 .…”

Macromolecular refinement of X-ray and cryoelectron microscopy structures with Phenix/OPLS3e for improved structure and ligand quality

“…The Phenix/OPLS3e implementation has already seen applications in cryo-EM-enabled SBDD of macrocycles in ribosomes (Li et al, 2020;Qi et al, 2019) and determining high-confidence ligand binding poses in ambiguous cryo-EM structures in the GemSpot pipeline (Robertson et al, 2020). We foresee our approach as an accessible and user-friendly implementation of a high-quality force field to improve structure and ligand quality, especially when using lower-resolution data provided either by ll Resource crystallography and cryo-EM, and as an additional measure for reducing overfitting in cryo-EM structure refinement and cryo-EM ligand fitting.…”