The XChem platform at Diamond XChem training – April 2019

References on XChem Crystal preparation: Collins, P. M., Douangamath, A., Talon, R., Dias, A., Brandao-Neto, J., Krojer, T., & Von Delft, F. (2018). Achieving a Good Crystal System for Crystallographic X-ray Fragment Screening. Preprints(2018090383). doi:10.20944/preprints201809.0383.v1 Crystal soaking: Collins, P. M., Ng, J. T., Talon, R., Nekrosiute, K., Krojer, T., Douangamath, A., . . . von Delft, F. (2017). Gentle, fast and effective crystal soaking by acoustic dispensing. Acta Crystallogr D Struct Biol, 73(Pt 3), 246-255. doi:10.1107/S205979831700331X Fragment library design: Cox, O. B., Krojer, T., Collins, P., Monteiro, O., Talon, R., Bradley, A., . . . Brennan, P. E. (2016). A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain. Chem Sci, 7(3), 2322-2330. doi:10.1039/c5sc03115j Data processing: Krojer, T., Talon, R., Pearce, N., Collins, P., Douangamath, A., Brandao-Neto, J., . . . von Delft, F. (2017). The XChemExplorer graphical workflow tool for routine or large-scale protein-ligand structure determination. Acta Crystallogr D Struct Biol, 73(Pt 3), 267-278. doi:10.1107/S2059798316020234 Fragment hit detection: Pearce, N. M., Krojer, T., Bradley, A. R., Collins, P., Nowak, R. P., Talon, R., . . . von Delft, F. (2017). A multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density. Nat Commun, 8, 15123. doi:10.1038/ncomms15123

XChem at Diamond Complete screening experiment: crystal-to-model Prepare 1000+ crystals Measure 1000+ crystals Analyse 1000+ datasets

Readout PNP2 enzyme from pyrimidine synthesis JMJD2D, a histone lysine demethylase As expected

XChem at Diamond Complete screening experiment: crystal-to-model Prepare 1000+ crystals Measure 1000+ crystals Analyse 1000+ datasets

XChem Fragment libraries Fragment library XChem Fragment libraries DSi-Poised Library (Diamond-SGC-iNext, ex DSPL, provided by Enamine) -768 fragments, in 500mM d6-dmso For all other libraries, please contact your local contact well ahead! Kidd3D Library (University of Cambridge/Pharmenable) -137 fragments, in 250- 500mM in d6-dmso-125. Contact Pharmenable ahead Leeds3D 125 fragments, in 100-500mM – Contact Adam Nelson ahead Covalent library – 993 fragments, in 20mM - Contact Nir London ahead Own libraries in labcyte-compatible plates https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/Fragment-Libraries.html

Poised fragments: cheap single-step expansion Poised reactions are: robust and reliable; make drug-like products; be possible using commercially available starting points; compatible with a range of substrates. Oakley Cox (Paul Brennan) Full paper: Cox et al, Chemical Science, 2016

Inverted crystallisation plate Fragment library plate Echo crystal soaking Inverted crystallisation plate Fragment library plate Acoustic droplet ejection Small volumes and fast Fine control over drop placement Random access dispensing (“cherry-picking”) Labcyte Echo Fragment library labcyte https://www.youtube.com/watch?v=tYg8DpYGIz0

Crystal soaking with the Echo Patrick Collins P. M. Collins et al. -  Acta Cryst. (2017). D73, 246-255 Compound/solvent diffusion across drop Crystallisation plate 2.5 nL Echo drops Requested Delivered Compound dispensing patterns

Rapid crystal soaking 900 crystal soaks in 30 min Click to target Jia Crystal targets Click to target X -307, y 365 Jia 900 crystal soaks in 30 min