Small Angle-X-ray Scattering Tensor Tomography (SAS-TT) is a relatively new but powerful technique for studying the multiscale architecture of hierarchical structures particularly relevant to life science applications. Currently, the technique is very demanding on synchrotron beamtime, which limits its applications, especially for cases requiring a statistically relevant number of samples. This study reports the first SAS-TT measurement at a macromolecular X-ray crystallography beamline, PX-I at the Swiss Light Source (SLS), with an improvement in acquisition time from 96 h/Mvoxel in the pilot experiments to 6 h/Mvoxel with comparable sampling, defining a new standard for fast SAS-TT with a micrometer beam size and allowing to record a full tomogram in 1.2 h. Measurements are performed on the long and lenticular process of the incus bone, one of the three human auditory ossicles. The main orientation and degree of alignment of the mineralised collagen fibrils are characterised, as well as the size and shape of the mineral particles which show relevant variations in different tissue locations. The study reveals three distinct regions of high fibril alignment, most likely important pathways of sound throughout the ossicular chain, and highlights the technique's potential to aid in future developments in middle ear reconstructive surgery.

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.

Supplementary Figure S1 describes mRNA and protein expression of PARG in shPARG models and in vitro sensitivity to oxaliplatin.

Supplementary Fig S7 describes western blot and RTPCR of PARG in modified shPARG cells.

Supplementary Table 1 describes BRCA and KRAS status of PDX models and IC50 of PDDX-001 and olaparib in PDAC/PDX lines

Over the last two decades, fragment-based drug discovery has emerged as an effective and efficient method to identify chemical scaffolds for the development of novel lead compounds.The inherent "start small -elaborate efficiently" approach allows issues like compound-selectivity, toxicity and efficiency to be addressed from a very early development state on, while saving time and resources compared to classical high throughput screening of larger compounds.X-ray crystallography has been used for a long time as an important orthogonal method for validating binders discovered by higher throughput screening methods and to elucidate corresponding ligandtarget interactions.Advances in beamline-and crystal-harvestinginstrumentation have tremendously increased the throughput of X-ray crystallography in the last few years.This has facilitated the establishment of macromolecular crystallography as a powerful primary screening method for the identification of ligand binding.Combining fragment screening with the high information content derived from crystal-based fragment screening results in a powerful platform for structure-based drug discovery.Here, we proudly present the Fast Fragment and Compound Screening pipeline (FFCS) at the Swiss Light Source (PSI, Switzerland) and its application in the development of novel drugs 1 .

Supplementary Figure S5 describes PARG ELISA.

Ions are important to modulate protein properties, including solubility and stability, through specific ion effects. Ionic liquids (ILs) are designer salts with versatile ion combinations with great potential to control protein properties. Although protein-ion binding of common metals is well-known, the IL effect on proteins is not well understood. Here, we employ the model protein lysozyme in dilute and concentrated IL solutions to determine the specific ion binding effect on protein phase behaviour, activity, size and conformational change, aggregation and intermolecular interactions. A combination of spectroscopic techniques, activity assays, small-angle X-ray scattering, and crystallography highlights that ILs, particularly their anions, bind to specific sites in the protein hydration layer via polar contacts on charged, polar and aromatic residues. The specific ion binding can induce more flexible loop regions in lysozyme, while the ion binding in the bulk phase can be more dynamic in solution. Overall, the protein behaviour in ILs depends on the net effect of nonspecific interactions and specific ion binding. Compared to formate, the nitrate anion induced high protein solubility, low activity, elongated shape and aggregation, which is largely owing to its higher propensity for ion binding. These findings provide new insights into protein-IL binding interactions and using ILs to modulate protein properties.

Supplementary Fig S4 describes synergy plots in Hs 766T cells.

Supplementary Figure S6 describes cleaved caspase 3 expression and quantitation of H2AX foci in MIA PaCa-2 after combination of PDDX-001 and oxaliplatin.

Supplementary Figure S3 describes synthetic lethality of olaparib with siDDR in PDAC.

CX-ASAP is a new open-source software project designed to greatly reduce the time required to analyse crystallographic data collected under varying conditions. Scripted in Python3, CX-ASAP can automatically refine, finalize and analyse data collections with wide-ranging temperatures, pressures etc. This is achieved using a reference structure, allowing for quick identification of problems, phase changes and even model comparison. The modular design means that new features and customized scripts can be easily added, tailoring the capabilities to the specific needs of the user. It is envisioned that CX-ASAP will help to close the growing gap between fast collection times and slow data finalization.

Supplementary Fig S2 describes protein expression of PARG across PDAC cell models and drug sensitivity of PDAC, KPC, DLD and RKO cells to olaparib and/or PDDX-001.

The solid-state phase transformation in nickel(II) bis-(diisopropyldithiocarbonate) is analyzed using a combination of highspeed in situ single-crystal diffraction, terahertz spectroscopy, optical microscopy, thermal analysis, and density functional theory. We show that the monoclinic P2(1)/c structure of this compound undergoes a displacive phase change at about 3 degrees C. The monoclinic angles and unit cell volumes change reversibly between 110.3 degrees/2265 angstrom(3) and 103.8 degrees/2168 angstrom(3). An analysis of atomic positions using high-resolution in situ synchrotron X-ray diffraction data revealed details of the atomic displacements that show a change in order that precedes and accompanies the change in structure. The structural changes are rapid and are manifested as reversible macroscale crystal movement and jumping (thermosalience) and represent the first case of thermosalience in dithiocarbamate complexes.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

The MERS coronavirus (MERS-CoV) is a highly pathogenic, emerging virus that produces accessory proteins to antagonize the host innate immune response. The MERS-CoV ORF4b protein has been shown to bind preferentially to the nuclear import adapter IMPα3 in infected cells, thereby inhibiting NF-κB-dependent innate immune responses. Here, we report high-resolution structures of ORF4b bound to two distinct IMPα family members. Each exhibit highly similar binding mechanisms that, in both cases, lack a prototypical Lys bound at their P2 site. Mutations within the NLS region dramatically alter the mechanism of binding, which reverts to the canonical P2 Lys binding mechanism. Mutational studies confirm that the novel binding mechanism is important for its nuclear import, IMPα interaction, and inhibition of innate immune signaling pathways. In parallel, we determined structures of the nuclear binding domain of NF-κB component p50 bound to both IMPα2 and α3, demonstrating that p50 overlaps with the ORF4b binding sites, suggesting a basis for inhibition. Our results provide a detailed structural basis that explains how a virus can target the IMPα nuclear import adapter to impair immunity, and illustrate how small mutations in ORF4b, like those found in closely related coronaviruses such as HKU5, change the IMPα binding mechanism.