An extensive study of error distributions for calculating hydrogen and carbon NMR chemical shifts at Hartree-Fock (HF), density functional theory (DFT), and Møller-Plesset second-order perturbation theory (MP2) levels is presented. Our investigation employs accurate CCSD(T)/cc-pVQZ calculations for providing reference data for 48 hydrogen and 40 carbon nuclei within an extended set of chemical compounds covering a broad range of the NMR scale with high relevance to chemical applications, especially in organic chemistry. Besides the approximations of HF, a variety of DFT functionals, and conventional MP2, we also present results with respect to a spin component-scaled MP2 (GIAO-SCS-MP2) approach. For each method, the accuracy is analyzed in detail for various basis sets, allowing identification of efficient combinations of method and basis set approximations.

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPR ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.