@article {68521, title = {From primordial clocks to circadian oscillators}, journal = {Nature}, year = {2023}, month = {2023 Apr 01}, abstract = {

Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in\ Rhodobacter sphaeroides\ (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS\ where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS\ and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 {\r A}. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

}, doi = {10.1038/s41586-023-05836-9}, url = {https://doi.org/10.1038/s41586-023-05836-9}, author = {Warintra Pitsawong and Ricardo A. P. P{\'a}dua and Timothy Grant and Marc Hoemberger and Renee Otten and Niels Bradshaw and Nikolaus Grigorieff and Dorothee Kern} } @article {68010, title = {In situ single particle classification reveals distinct 60S maturation intermediates in cells.}, journal = {eLife}, year = {2022}, month = {2022 Aug 25}, abstract = {

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure.\ In situ\ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of\ in situ\ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the\ Saccharomyces cerevisiae\ cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

}, doi = {10.7554/eLife.79272}, url = {https://doi.org/10.7554/eLife.79272}, author = {Bronwyn A. Lucas and Kexin Zhang and Sarah Loerch and Nikolaus Grigorieff} }