# Enhancing the Outcome of Crystallographic Fragment Screening by Choosing the Optimal Protein Crystal Form

## TL;DR

Choosing the right protein crystal form can greatly improve the success rate of drug discovery screening.

## Contribution

The study shows that crystal form significantly affects hit rates in crystallographic fragment screening.

## Key findings

- Orthorhombic crystals of SARS-CoV-2 protease had a 16% hit rate compared to 3% in monoclinic crystals.
- Larger solvent channels in orthorhombic crystals correlate with higher hit rates in fragment screening.

## Abstract

Improving health and quality of life in our society is a key focus of drug development. Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines. Crystallographic fragment screening (CFS) is increasingly being employed as an early screening method in drug discovery projects. Here, we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates. Two CFS campaigns are carried out against the two crystal forms of the SARS‐CoV‐2 main protease, using the same fragment library and an almost identical experimental setup. Although both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns differ significantly. A hit rate of 3% is determined for the monoclinic crystals, while a hit rate of 16% is observed for the orthorhombic crystals. These findings are consistent with the more open molecular packing in the orthorhombic crystals, where the solvent channels leading to the active sites are approximately twice the size of those in the monoclinic crystal form. Our results highlight the critical importance of the crystal form in a crystallographic screening, identifying it as one of the most important parameters to optimize when preparing a CFS campaign.

The selection of the crystal form impacts crystallographic fragment screening (CFS) hit rates. Almost identical CFS campaigns using monoclinic versus orthorhombic crystals of SARS‐CoV‐2 main protease yield hit rates of 3% and 16%. The higher hit rate correlates with larger solvent channels and a less constrained active site in the orthorhombic form.© 2026 WILEY‐VCH GmbH