Time-resolved cryo-EM is an emerging technique in structural biology that allows the user to capture structural states which would otherwise be too transient for standard methods. There has been a resurgence in technical advancements in this field in the last five years and this review provides a summary of the technical highlights.

It is found that research mentoring of undergraduate students can result in meaningful contributions to scientific disciplines. Here, an outcome-oriented, skill-based mentoring strategy is proposed and evaluated in the context of the CASP14 community challenge.

Analysis of high-resolution data indicated that X-ray damage has little impact on conformational ensemble information from protein crystals at room temperature (277 K). In contrast, X-ray damage can alter conformational distributions obtained from cryo-cooled crystals under common experimental conditions, complicating structural interpretations in some instances. The results presented provide a strong motivation for the expanded use of room-temperature X-ray crystallography.

A room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed and applied to study ligand binding and protein plasticity.

The structure of the full-length Drosophila cryptochrome as solved by fixed-target serial synchrotron crystallography at the Cornell High Energy Synchrotron Source is presented. Scaling and filtering methods were performed to improve the overall quality of the final model.

Room-temperature crystallography enables researchers to resolve the conformational heterogeneity of structures. Here, the native SAD phasing of four structures at 295 K highlights the strengths of room-temperature diffraction experiments, including detailed anomalous difference maps and alternate conformations that are well supported by the electron density.

Protein crystals grown in microfluidic droplets are shown to be an effective and robust platform for storage, transport and serial crystallography data collection using a silicon fixed-target serial device with a minimal impact on diffraction quality.

Accurate electrostatic potential maps and electron-density maps of proteins are calculated based on the transferable aspherical atom model using a pseudoatom data bank and are compared with the experimental data.

Multiple approaches to data merging and scaling and anomalous substructure determination were used to solve the structure of a large asymmetric unit from weakly diffracting crystals by sulfur SAD.

Database-derived water probability densities around structurally and sequentially distinct DNA dinucleotide fragments reproduce the known hydration motifs, which thus can be used as building blocks to predict DNA hydration.

A new approach for simulating contrast-variation small-angle scattering experiments is presented.

The 3 Å resolution crystal structure of the Pseudomonas aeruginosa virulence factor CbpD both supports and challenges the current model of how lytic polysaccharide monooxygenases bind chitin and raises interesting possibilities about how type 2 secretion-system substrates may interact with the secretion machinery. This structure also demonstrates the utility of new, AI-powered, protein structure-prediction algorithms in making challenging structural targets tractable.