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To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach. [ABSTRACT FROM AUTHOR]

Within the structural biology field, X-ray crystallography prevails as the dominant technique to determine the structures of macromolecules, producing, as of November 2020, more than 150,000 structures since its inception (https://www.rcsb.org/). New technologies developed at X-ray free electron lasers (XFELs) and the birth of serial femtosecond crystallography (SFX) have opened up a new era in structural biology. The review article by Cheng [[2]] is a comprehensive review that covers most of the current and future trends on sample delivery methods for SFX at XFELs. [Extracted from the article]

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]