CryoSTAR: leveraging structural priors and constraints for cryo-EM heterogeneous reconstruction.

Resolving conformational heterogeneity in cryogenic electron microscopy datasets remains an important challenge in structural biology. Previous methods have often been restricted to working exclusively on volumetric densities, neglecting the potential of incorporating any preexisting structural knowledge as prior or constraints. Here we present cryoSTAR, which harnesses atomic model information as structural regularization to elucidate such heterogeneity.

Cloud computing platforms to support cryo-EM structure determination.

Leveraging the power of single-particle cryo-electron microscopy (cryo-EM) requires robust and accessible computational infrastructure. Here, we summarize the cloud computing landscape and picture the outlook of a hybrid cryo-EM computing workflow, and make suggestions to the community to facilitate a future for cryo-EM that integrates into cloud computing infrastructure.

High-resolution cryo-EM using beam-image shift at 200 keV.

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed.

High-Throughput Cryo-EM Enabled by User-Free Preprocessing Routines.

Single-particle cryoelectron microscopy (cryo-EM) continues to grow into a mainstream structural biology technique. Recent developments in data collection strategies alongside new sample preparation devices herald a future where users will collect multiple datasets per microscope session. To make cryo-EM data processing more automatic and user-friendly, we have developed an automatic pipeline for cryo-EM data preprocessing and assessment using a combination of deep-learning and image-analysis tools.

SMALL-LABS: Measuring Single-Molecule Intensity and Position in Obscuring Backgrounds.

Single-molecule and super-resolution imaging relies on successful, sensitive, and accurate detection of the emission from fluorescent molecules. Yet, despite the widespread adoption of super-resolution microscopies, single-molecule data processing algorithms can fail to provide accurate measurements of the brightness and position of molecules in the presence of backgrounds that fluctuate significantly over time and space. Thus, samples or experiments that include obscuring backgrounds can severely, or even completely, hinder this process.

Dynamic Exchange of Two Essential DNA Polymerases during Replication and after Fork Arrest.

The replisome is a multiprotein machine responsible for the faithful replication of chromosomal and plasmid DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in live Bacillus subtilis cells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following replication fork stress using damage-independent and damage-dependent conditions.

Visualizing bacterial DNA replication and repair with molecular resolution.

Although DNA replication and repair in bacteria have been extensively studied for many decades, in recent years the development of single-molecule microscopy has provided a new perspective on these fundamental processes. Because single-molecule imaging super-resolves the nanometer-scale dynamics of molecules, and because single-molecule imaging is sensitive to heterogeneities within a sample, this nanoscopic microscopy technique measures the motions, localizations, and interactions of proteins in real time without averaging ensemble observations, both in vitro and in vivo. In this Review, we provide an overview of several recent single-molecule fluorescence microscopy studies on DNA replication and repair.

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells.

PolC is one of two essential replicative DNA polymerases found in the Gram-positive bacterium Bacillus subtilis. The B. subtilis replisome is eukaryotic-like in that it relies on a two DNA polymerase system for chromosomal replication.