The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system.

All widely used mRNA vaccines against COVID-19 contain in their sequence 1-methylpseudouridine (m1 Psi) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 & Aring;, respectively) of one such double-stranded 12-mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the X-ray structure of 1-methyl-pseudouridine itself was determined.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris , as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

Crystallographic fragment screening, which involves screening small-molecule libraries against crystals of a target protein, is an essential tool in modern drug discovery.The technique relies on the high-throughput generation of cryo-cooled, soaked crystals followed by fast and efficient data collection at a synchrotron beamline.Each campaign may generate hundreds or thousands of samples, and the most efficient strategy for acquiring data is to use unattended data collection followed by automatic data processing.The macromolecular crystallography (MX) group at the Swiss Light Source operates a fast fragment and compound screening (FFCS) pipeline that uses Smart Digital User (SDU) software to collect data at the beamlines.This presentation will give an overview of SDU, describe how it has been implemented at the MX beamlines and present some recent case studies.Figure 1.Screenshot of SDU in action at beamline X06SA

The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.

Raw diffraction data for mpro-x1187 / PDB ID 5RFA (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RFA) - SARS-CoV-2 main protease in complex with Z2643472210 (SMILES:CN1C=CC(=N1)C(=O)NC[C@@H]2CCCO2) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x1374 / PDB ID 5RFM (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RFM) - SARS-CoV-2 main protease in complex with PCM-0102539 (SMILES:Cc1ccc(cc1)N(C2CS(=O)(=O)C=C2)C(=O)CCl) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0749 / PDB ID 5REN (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5REN) - SARS-CoV-2 main protease in complex with PCM-0102425 (SMILES:ClCC(=O)N1CCCC(C1)c2nc3ccccc3s2) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0305 / PDB ID 5R82 (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5R82) - SARS-CoV-2 main protease in complex with Z219104216 (SMILES:CCNC=1C=CC(C#N)=CN1) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x1336 / PDB ID 5RFI (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RFI) - SARS-CoV-2 main protease in complex with PCM-0102353 (SMILES:Cc1ccc(C)c(c1)S(=O)(=O)N2CCN(CC2)C(=O)CCl) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0769 / PDB ID 5RES (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RES) - SARS-CoV-2 main protease in complex with PCM-0102281 (SMILES:Fc1ccccc1S(=O)(=O)N2CCN(CC2)C(=O)CCl) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0752 / PDB ID 5REO (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5REO) - SARS-CoV-2 main protease in complex with PCM-0102578 (SMILES:ClCC(=O)NCc1ccc2OCOc2c1) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x1226 / PDB ID 5RFB (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RFB) - SARS-CoV-2 main protease in complex with Z1271660837 (SMILES:CCNCC1=CN(C)N=N1) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0376 / PDB ID 5REA (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5REA) - SARS-CoV-2 main protease in complex with Z31432226 (SMILES:O=C(N1CCCCCC1)C=2C=CC=3OCOC3C2) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x1119 / PDB ID 5RF8 (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RF8) - SARS-CoV-2 main protease in complex with Z271004858 (SMILES:NC=1C=CC(=CC1)S(=O)(=O)NC=2C=CC=CN2) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Raw diffraction data for mpro-x0194 / PDB ID 5RE6 (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RE6) - SARS-CoV-2 main protease in complex with Z54571979 (SMILES:CC(=O)NC=1C=CC(OC=2N=CC=CN2)=CC1) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10-14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.

Raw diffraction data for mpro-x1375 / PDB ID 5RFN (see: https://www.ebi.ac.uk/pdbe/entry/pdb/5RFN) - SARS-CoV-2 main protease in complex with PCM-0102868 (SMILES:Fc1ccc(cc1)N(C2CS(=O)(=O)C=C2)C(=O)CCl) collected as part of an XChem crystallographic fragment screening campaign on beamline i04-1 at Diamond Light Source. The deposited structure was automatically processed with standard Diamond tools and PanDDA, however the raw data are being made available to allow reanalysis by any interested party. For more details see: https://www.diamond.ac.uk/covid-19/for-scientists/Main-protease-structure-and-XChem.html