The integrative role of cryo electron microscopy in molecular and cellular structural biology.

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo-EM) is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This 'revolution in resolution' is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo-EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo-EM in synergy with other methods such as X-ray crystallography, fluorescence imaging or focussed-ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi-scale and multi-resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.

Perturbations in nucleosome structure from heavy metal association.

Heavy metals have the potential to engage in strong bonding interactions and can thus function in essential as well as toxic or therapeutic capacities. We conducted crystallographic analyses of heavy cation binding to the nucleosome core particle and found that Co(2+) and Ni(2+) preferentially associate with the DNA major groove, in a sequence- and conformation-dependent manner. Conversely, Rb(+) and Cs(+) are found to bind only opportunistically to minor groove elements of the DNA, in particular at narrow AT dinucleotide sites. Furthermore, relative to Mn(2+) the aggressive coordination of Co(2+) and Ni(2+) to guanine bases is observed to induce a shift in histone-DNA register around the nucleosome center by stabilizing DNA stretching over one region accompanied by expulsion of two bases at an opposing location. These 'softer' transition metals also associate with multiple histone protein sites, including inter-nucleosomal cross-linking, and display a proclivity for coordination to histidine. Sustained binding and the ability to induce structural perturbations at specific locations in the nucleosome may contribute to genetic and epigenetic mechanisms of carcinogenesis mediated by Co(2+) and Ni(2+).

Structural insight into the sequence dependence of nucleosome positioning.

Nucleosome positioning displays sequence dependency and contributes to genomic regulation in a site-specific manner. We solved the structures of nucleosome core particle composed of strong positioning TTTAA elements flanking the nucleosome center. The positioning strength of the super flexible TA dinucleotide is consistent with its observed central location within minor groove inward regions, where it can contribute maximally to energetically challenging minor groove bending, kinking and compression. The marked preference for TTTAA and positioning power of the site 1.5 double helix turns from the nucleosome center relates to a unique histone protein motif at this location, which enforces a sustained, extremely narrow minor groove via a hydrophobic "sugar clamp." Our analysis sheds light on the basis of nucleosome positioning and indicates that the histone octamer has evolved not to fully minimize sequence discrimination in DNA binding.