Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Generally, protein fusions with labelling enzymes are transiently overexpressed to perform these experiments. Using a pipeline for the rapid generation CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. We found TurboID and its shorter variant miniTurboID to be superior above other labelling enzymes at physiological expression levels. Endogenous tagging of the μ subunit of the AP-1 complex increased the sensitivity for detection of interactors in a proximity labelling experiment and resulted in a more comprehensive mass spectrometry data set. We were able to identify several known interactors of the complex and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry data in just over a month.

Crystallographic fragment screening (CFS) has become one of the major techniques for screening compounds in the early stages of drug-discovery projects. Following the advances in automation and throughput at modern macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to organizing and handling the analysis of such projects. The complexity that emerges from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on the scientific questions instead of on software technicalities. FragMAXapp is the fragment-screening project-management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from the powerful computing infrastructure of large-scale facilities and, as a web application, it is accessible from everywhere.

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