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Ever since the resolution revolution in 2013, cryo-electron microscopy (cryo-EM) has become a powerful methodology in structural biology that is especially suited to study the structure of large flexible molecular complexes. Since then, the need of setting up state-of-the-art cryo-EM facilities around the world has increased tremendously. Access to high-end cryo-EM instrumentation is however expensive and requires expertise. The establishment of large cryo-EM centers worldwide, many of which provide academic users free access for both data collection and user training, has been possible with the support of government agencies across the globe. In addition, many universities, and private institutions like the Van Andel Institute (VAI) have made significant investments to establish their own cryo-EM core facilities, ensuring on-site access to their researchers. This paper aims to serve as a blueprint for establishing a new mid-sized cryo-EM facility, as it provides key information based on our experience at VAI and discusses strategies used to optimize routine operation towards high performance and efficiency for single-particle cryo-EM. Information regarding initial planning, selection of equipment as well as the development of IT solutions that were required to improve data collection and analysis are included. In addition, we present an account of the most common issues affecting operation as well as the needs for maintenance over a 6-year period, which can help interested parties to estimate the long-term costs of running this type of facility. Lastly, a brief discussion on the pros and cons of establishing the facility is also included., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Meng, Ratnayake, Escobar Galvis, Kotecki, Ramjan and Zhao.)

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed partially methylated domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using whole-genome bisulfite sequencing (WGBS) and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context ('solo-WCGWs') identified previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increased with age, beginning during fetal development, and appeared to track the accumulation of cell divisions. In cancer, PMD hypomethylation depth correlated with somatic mutation density and cell cycle gene expression, consistent with its reflection of mitotic history and suggesting its application as a mitotic clock. We propose that late replication leads to lifelong progressive methylation loss, which acts as a biomarker for cellular aging and which may contribute to oncogenesis.

We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol "protein scanning with virtual ligand screening" (PSVLS). Hit selection used an in-house developed holistic binding scoring method. Selected hits were tested experimentally for inhibitory activity using fluorescence resonance energy transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex through either radiolabeled ligand binding assays (nonfluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A's proteolytic activity experimentally with an IC50 of 5.2 μM. A fluorescent derivative was also confirmed to bind to the toxin and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multiprobe detection assays.

Extensive changes in DNA methylation are common in cancer and may contribute to oncogenesis through transcriptional silencing of tumor-suppressor genes. Genome-scale studies have yielded important insights into these changes but have focused on CpG islands or gene promoters. We used whole-genome bisulfite sequencing (bisulfite-seq) to comprehensively profile a primary human colorectal tumor and adjacent normal colon tissue at single-basepair resolution. Regions of focal hypermethylation in the tumor were located primarily at CpG islands and were concentrated within regions of long-range (>100 kb) hypomethylation. These hypomethylated domains covered nearly half of the genome and coincided with late replication and attachment to the nuclear lamina in human cell lines. We confirmed the confluence of hypermethylation and hypomethylation within these domains in 25 diverse colorectal tumors and matched adjacent tissue. We propose that widespread DNA methylation changes in cancer are linked to silencing programs orchestrated by the three-dimensional organization of chromatin within the nucleus.

Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing gamma-core structural domains. The C-terminal alpha-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic alpha-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments.