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The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R -factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 Å. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 Å is predicted at 200 keV if MR can be used, using R -factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is an enzyme primarily known for the production of lysophosphatidic acid. Being involved in the development of major human diseases, such as cancer and neurodegenerative diseases, the enzyme has been featured in multiple studies as a pharmacological target. We previously found that the cannabinoid tetrahydrocannabinol (THC) could bind and act as an excellent inhibitor of ATX. This study aims to use the cannabinoid scaffold as a starting point to find cannabinoid-unrelated ATX inhibitors, following a funnel down approach in which large chemical libraries sharing chemical similarities with THC were screened to identify lead scaffold types for optimization. This approach allowed us to identify compounds bearing chromone and indole scaffolds as promising ATX inhibitors. Further optimization led to MEY-003, which is characterized by the direct linkage of an N -pentyl indole to the 5,7-dihydroxychromone moiety. This molecule has potent inhibitory activity towards ATX-β and ATX-ɣ as evidenced by enzymatic studies and its mode of action was rationalized by structural biology studies using macromolecular X-ray crystallography. [Display omitted] • Autotaxin (ATX) is an enzyme known for the production of lysophosphatidic acid (LPA) an important signaling lipid • Generation of new chromone-derived inhibitors of Autotaxin, a therapeutic target for cancer and neurodegenerative diseases. • Chromones linked to an indole moiety identified as the most active inhibitors using biochemical and cellular assays. • The most active compound (MEY-003) showed potent inhibitory activity towards ATX-β and ATX-ɣ in the nanomolar range. • The ATX binding mode of MEY-003 was experimentally determined using X-ray crystallography. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia. • X-ray free-electron laser unveils alternative protein structure of lipoprotein • Advanced molecular dynamics explore conformational space among solved structures • Virtual ligand screening shows possible lead fragments for potential drug therapies Using X-ray free-electron laser (XFEL) technology, Zook et al. describe an alternative protein conformation of the virulence determinant Flpp3 and use molecular dynamics (MD) to investigate conformational landscapes between these conformations. MD identifies several intermediate conformations that allow these conformational transitions to occur at physiologically relevant temperatures and provide insight to potential future drug therapies for the potentially deadly disease tularemia. [ABSTRACT FROM AUTHOR]