Fragment Screen and Data Analysis - StructuralGenomicsConsortium/CNP25-CHIKV-nsP3-Macrodomain GitHub Wiki

The crystallographic fragment screen against the CHIKV nsP3 macrodomain was carried out by Jasmin Aschenbrenner (XChem) at Diamond Light Source, UK.

We screened 1385 fragments from the DSi-Poised Library, Minifrags, Fraglites, Peplites, York 3D, SpotXplorer, and Essential Fragment Library (Enamine). Fragments were soaked into drops containing CHIKV Mac crystals at 25 % (v/v) using acoustic dispensing with an Echo 650 liquid handler (Labcyte). After incubation for 1-3 h at 20°C, crystals were harvested and cryo-cooled in liquid nitrogen without additional cryoprotectant.

Data was collected at the I04-1 beamline (Diamond Light Source, UK) at 100 K and automatically processed with Diamond Light Source’s autoprocessing pipelines using XDS and either xia2, autoPROC or DIALS with the default settings.

Analysis of fragment binding was done using the XChemExplorer. Electron density maps were generated with Dimple, ligand restraints were calculated with GRADE and ligand-binding events were identified using PanDDA2 by @ConorFWild. Ligands were modelled into PanDDA2-calculated event maps using its autobuild function or manually using Coot, and structures were refined by ensemble-refinement of ligand-free and ligand-bound states using Refmac.

Coordinates, structure factors and PanDDA2 event maps for the ground-state and 109 ligand-bound CHIKV Mac structures are deposited in the Protein Data Bank (group deposition G_1002294) with the following accession codes: 7H6J, 7H6K, 7H6L, 7H6M, 7H6N, 7H6O, 7H6P, 7H6Q, 7H6R, 7H6S, 7H6T, 7H6U, 7H6V, 7H6W, 7H6X, 7H6Y, 7H6Z, 7H70, 7H71, 7H72, 7H73, 7H74, 7H75, 7H76, 7H77, 7H78, 7H79, 7H7A, 7H7B, 7H7C, 7H7D, 7H7E, 7H7F, 7H7G, 7H7H, 7H7I, 7H7J, 7H7K, 7H7L, 7H7M, 7H7N, 7H7O, 7H7P, 7H7Q, 7H7R, 7H7S, 7H7T, 7H7U, 7H7V, 7H7W, 7H7X, 7H7Y, 7H7Z, 7H80, 7H81, 7H82, 7H83, 7H84, 7H85, 7H86, 7H87, 7H88, 7H89, 7H8A, 7H8B, 7H8C, 7H8D, 7H8E, 7H8F, 7H8G, 7H8H, 7H8I, 7H8J, 7H8K, 7H8L, 7H8M, 7H8N, 7H8O, 7H8P, 7H8Q, 7H8R, 7H8S, 7H8T, 7H8U, 7H8V, 7H8W, 7H8X, 7H8Y, 7H8Z, 7H90, 7H91, 7H92, 7H93, 7H94, 7H95, 7H96, 7H97, 7H98, 7H99, 7H9A, 7H9B, 7H9C, 7H9D, 7H9E, 7H9F, 7H9G, 7H9H, 7H9I, 7H9J. Additionally, structures have been made available on Fragalysis.

• Datasets successfully collected and processed: 1082 datasets
• Hit identification: 109 fragment hits (66 in the ADPr-binding site, 43 in sub-sites close to the ADPr-binding site)
• PoseButcher site analysis: 1- Ribose-binding site: 42 observations 2- Phosphate-binding site: 26 observations 3-Oxyanion site: 61 observations 4- Adenine site: 188 observations