High-Throughput Crystallography: Reliable and Efficient Identification of Fragment Hits

Abstract

Today the identification of lead structures for drug development often starts from small fragment-like molecules raising the chances to find compounds that successfully pass clinical trials. At the heart of the screening for fragments binding to a specific target, crystallography delivers structural information essential for subsequent drug design. While it is common to search for bound ligands in electron densities calculated directly after an initial refinement cycle, we raise the important question whether this strategy is viable for fragments characterized by low affinities. Here, we describe and provide a collection of high-quality diffraction data obtained from 364 protein crystals treated with diverse fragments. Subsequent data analysis showed that ∼25% of all hits would have been missed without further refining the resulting structures. To enable fast and reliable hit identification, we have designed an automated refinement pipeline that will inspire the development of optimized tools facilitating the successful application of fragment-based methods.

Intriguing role of water in protein-ligand binding studied by neutron crystallography on trypsin complexes

“…When lowering the temperature to 100 K during X-ray diffraction experiments, W2 was found to be rather H-bonded to W1 than to W3 (occupancies of 75 and 25% in our structure with PDB-code 5MNE). To analyze whether this temperature effect is relevant, we collected three additional X-ray diffraction data sets from non-deuterated uncomplexed trypsin crystals each at 295 and 100 K. All structures were equally processed via an automated refinement pipeline 20 , which places one W2 molecule into the center of the corresponding electron density feature. The resulting structures confirmed that the water pattern in the S 1 pocket differs at the two temperatures with W2 being much closer (by 0.4 Å) to W1 at 100 K (Fig.…”

“…c Temperature-dependent change in the Asp189 solvation pattern. Four individual X-ray structures of uncomplexed trypsin at cryogenic temperature that were determined via an automated refinement pipeline are shown in blue 20 . The data for these structures have been collected from one deuterated and three non-deuterated crystals.…”

“…Data collection statistics are given in Supplementary Tables 5 (100 K) and 6 (295 K). For a closer investigation of the water structure in the S 1 pocket of uncomplexed trypsin, we also collected three additional X-ray diffraction data sets from non-deuterated uncomplexed trypsin crystals each at 295 K and at 100 K. Data were processed and structures refined using our PHENIX-based 37 automated pipeline tool 20 . The crystal of trypsin in complex with the D -Phe-Pro analog of N -amidinopiperidine underwent X-ray exposure on beamline 14.1 at BESSY (Berlin, Germany) with subsequent XDS-based data processing.…”

“…Our previously published automated refinement pipeline script mentioned in the above experimental section is available from the corresponding author upon request 20 .…”

Protein Data Bank Japan (PDBj): updated user interfaces, resource description framework, analysis tools for large structures

“…Another recent trend in structural biology focuses on complex structures between proteins or between proteins and chemical compounds (25). To support analyses and modeling of complex structures, we have added HOMCOS (26), a tool for searching, analyzing and modeling of complex structures based on structural similarities.…”