The products of alginate degradation, alginate oligosaccharides (AOS), have potential applications in many areas, including functional foods and marine drugs. Enzyme-based approaches using alginate lyases have advantages in the preparation of well defined AOS and have attracted much attention in recent years. However, a lack of structural insight into the whole substrate-binding cleft for most known alginate lyases severely hampers their application in the industrial generation of well defined AOS. To solve this issue, AlyF was co-crystallized with the long alginate oligosaccharide G6 (L-hexaguluronic acid hexasodium salt), which is the longest bound substrate in all solved alginate lyase complex structures. AlyF formed interactions with G6 from subsites -3 to +3 without additional substrate-binding site interactions, suggesting that the substrate-binding cleft of AlyF was fully occupied by six sugars, which was further confirmed by isothermal titration calorimetry and differential scanning calorimetry analyses. More importantly, a combination of structural comparisons and mutagenetic analyses determined that three key loops (loop 1, Lys215-Glu236; loop 2, Gln402-Ile416; loop 3, Arg334-Gly348) mainly function in binding long substrates (degree of polymerization of >4). The potential flexibility of loop 1 and loop 2 might enable the substrate to continue to enter the cleft after binding to subsites +1 to +3; loop 3 stabilizes and orients the substrate at subsites -2 and -3. Taken together, these results provide the first possible alginate lyase-substrate binding profile for long-chain alginates, facilitating the rational design of new enzymes for industrial purposes.PII proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via PII proteins is modulated by effector molecules and post-translational modifications in the PII T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the PII protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the PII-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.Microcrystal electron diffraction (MicroED) has recently emerged as a promising method for macromolecular structure determination in structural biology. Since the first protein structure was determined in 2013, the method has been evolving rapidly. Several protein structures have been determined and various studies indicate that MicroED is capable of (i) revealing atomic structures with charges, (ii) solving new protein structures by molecular replacement, (iii) visualizing ligand-binding interactions and (iv) determining membrane-protein structures from microcrystals embedded in lipidic mesophases. However, further development and optimization is required to make MicroED experiments more accurate and more accessible to the structural biology community. Here, we provide an overview of the current status of the field, and highlight the ongoing development, to provide an indication of where the field may be going in the coming years. We anticipate that MicroED will become a robust method for macromolecular structure determination, complementing existing methods in structural biology.Synchrotron serial crystallography (SSX) is an emerging data-collection method for micro-crystallography on synchrotron macromolecular (MX) crystallography beamlines. At SPring-8, the feasibility of the fixed-target approach was examined by collecting data using a 2D raster scan combined with goniometer rotation. ZSH-2208 in vivo Results at cryogenic temperatures demonstrated that rotation is effective for efficient data collection in SSX and the method was named serial synchrotron rotation crystallography (SS-ROX). To use this method for room-temperature (RT) data collection, a humid air and glue-coating (HAG) method was developed in which data were collected from polyvinyl alcohol-coated microcrystals fixed on a loop under humidity-controlled air. The performance and the RT data-collection strategy for micro-crystallography were evaluated using microcrystals of lysozyme. Although a change in unit-cell dimensions of up to 1% was observed during data collection, the impact on data quality was marginal. A comparison of data obtained at various absorbed doses revealed that absorbed doses of up to 210 kGy were tolerable in both global and local damage. Although this limits the number of photons deposited on each crystal, increasing the number of merged images improved the resolution. On the basis of these results, an equation was proposed that relates the achievable resolution to the total photon flux used to obtain a data set.Methotrexate (MTX) is an anticancer and anti-rheumatoid arthritis drug that is considered to block nucleotide synthesis and the cell cycle mainly by inhibiting the activity of dihydrofolate reductase (DHFR). Using affinity-matrix technology and X-ray analysis, the present study shows that MTX also interacts with macrophage migration inhibitory factor (MIF). Fragment molecular-orbital calculations quantified the interaction between MTX and MIF based on the structure of the complex and revealed the amino acids that are effective in the interaction of MTX and MIF. It should be possible to design new small-molecule compounds that have strong inhibitory activity towards both MIF and DHFR by structure-based drug discovery.