Combining per-particle cryo-ET and cryo-EM single particle analysis to elucidate heterogeneous DNA-protein organization.

Cryo-electron microscopy single particle analysis (cryo-EM SPA) and cryo-electron tomography (cryo-ET) have historically been employed as distinct approaches for investigating molecular structures of disparate sample types, focusing on highly purified biological macromolecules and in situ cellular contexts, respectively. However, these techniques offer inherently complementary structural insights that, when combined, provide a more comprehensive understanding of complex biological systems. For example, if both techniques are applied to the same purified biological macromolecules, cryo-ET has the ability to resolve highly flexible yet strong signal features on an individual target molecule which will not be preserved in the high-resolution cryo-EM SPA results. In this review, we highlight recent achievements utilizing such applications to unveil new insights into the chromatin assembly and activities of DNA-protein assemblies. This convergence of cryo-EM SPA and cryo-ET holds great promise for elucidating new structural aspects of these essential molecular processes.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography.

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.