Summary A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

Time-resolved structural studies are becoming an important tool in understanding biological function. Here we describe a cryo-EM grid freezing device capable of rapidly mixing and plunge freezing grids within 10 ms.

Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 Å reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of ~10 ms, providing greater temporal resolution than previously reported approaches.

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Prodiginines are a group of naturally occurring pyrrole alkaloids produced by various microorganisms and known for their broad biological activities. The production of nature-inspired cyclic prodiginines was enabled by combining organic synthesis with a mutasynthesis approach based on the GRAS (generally recognized as safe) certified host strain Pseudomonas putida KT2440. The newly prepared prodiginines exerted antimicrobial effects against relevant alternative biotechnological microbial hosts whereas P. putida itself exhibited remarkable tolerance against all tested prodiginines, thus corroborating the bacterium's exceptional suitability as a mutasynthesis host for the production of these cytotoxic secondary metabolites. Moreover, the produced cyclic prodiginines proved to be autophagy modulators in human breast cancer cells. One promising cyclic prodiginine derivative stood out, being twice as potent as prodigiosin, the most prominent member of the prodiginine family, and its synthetic derivative obatoclax mesylate.

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

Bacterial metabolites represent an invaluable source of bioactive molecules which can be used as such or serve as chemical frameworks for developing new antimicrobial compounds for various applications including crop protection against pathogens. Prodiginines are tripyrrolic, red-colored compounds produced by many bacterial species. Recently, due to the use of chemical-, bio-, or mutasynthesis, a novel group of prodiginines was generated. In our study, we perform different assays to evaluate the effects of prodigiosin and five derivatives on nematodes and plant pathogenic fungi as well as on plant development. Our results showed that prodigiosin and the derivatives were active against the bacterial feeding nematode Caenorhabditis elegans in a concentration- and derivative-dependent manner while a direct effect on infective juveniles of the plant parasitic nematode Heterodera schachtii was observed for prodigiosin only. All compounds were found to be active against the plant pathogenic fungi Phoma lingam and Sclerotinia sclerotiorum. Efficacy varied depending on compound concentration and chemical structure. We observed that prodigiosin ( 1 ), the 12 ring- 9 , and hexenol 10 derivatives are neutral or even positive for growth of Arabidopsis thaliana depending on the applied compound concentration, whereas other derivatives appear to be suppressive. Our infection assays revealed that the total number of developed H. schachtii individuals on A. thaliana was decreased to 50% in the presence of compounds 1 or 9 . Furthermore, female nematodes and their associated syncytia were smaller in size. Prodiginines seem to indirectly inhibit H. schachtii parasitism of the plant. Further research is needed to elucidate their mode of action. Our results indicate that prodiginines are promising metabolites that have the potential to be developed into novel antinematodal and antifungal agents.

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