A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F1-ATPase data set and a 4.5 Å resolution SecYEG–SecA complex data set. All of the models were automatically built by the method to Rfree values of between 28.9 and 39.9% and were free from the initial model bias

A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F_1-ATPase data set and a 4.5 Å resolution SecYEG–SecA complex data set. All of the models were automatically built by the method to R_(free) values of between 28.9 and 39.9% and were free from the initial model bias

<i>In vitro</i> antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

BackgroundCurrent approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.MethodsSARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.ResultsDaclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.ConclusionsDaclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy

Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS

AbstractGrowth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras·GTP. The crystal structures suggest that Ras·GTP stabilizes the active site of SOS allosterically, and we show that Ras·GTP forms ternary complexes with SOScat in solution and increases significantly the rate of SOScat-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras

Architecture of the linker-scaffold in the nuclear pore

Quantitative docking of crystal and single particle cryo-EM structures into low resolution human and S. cerevisiae cryo-ET and X. laevis cryo-EM maps of the nuclear pore complex.Related Publication: Architecture of the linker-scaffold in the nuclear pore Petrovic, Stefan Caltech Samanta, Dipanjan Caltech Perriches, Thibaud Caltech Bley, Christopher Caltech Thierbach, Karsten Caltech Brown, Bonnie Caltech Nie, Si Caltech Mobbs, George Caltech Stevens, Taylor Caltech Liu, Xiaoyu Caltech Tomaleri, Giovani Pinton Caltech Schaus, Lucas Caltech Hoelz, Andre Caltech Science 2022-06-10 https://doi.org/10.1126/science.abm9798 en

Architecture of the cytoplasmic face of the nuclear pore

Quantitative docking of crystal structures into low resolution human cryo-ET and X. laevis cryo-EM maps of the nuclear pore complex cytoplasmic face.Related Publication: Architecture of the cytoplasmic face of the nuclear pore Bley, Christopher Caltech Nie, Si Caltech Mobbs, George Caltech Petrovic, Stefan Caltech Gres, Anna Caltech Liu, Xiaoyu Caltech Mukherjee, Somnath The University of Chicago Harvey, Sho Caltech Huber, Ferdinand Caltech Lin, Daniel Caltech Brown, Bonnie Caltech Tang, Aaron Caltech Rundlet, Emily Caltech Correia, Ana Caltech Chen, Shane National Institute of Child Health and Human Development Regmi, Saroj National Institute of Child Health and Human Development Jette, Claudia Caltech Stevens, Taylor Caltech Dasso, Mary National Institute of Child Health and Human Development Patke, Alina Caltech Palazzo, Alexander University of Toronto Kossiakoff, Anthony The University of Chicago Hoelz, Andre Caltech Science 2022-06-10 https://doi.org/10.1126/science.abm9129 en

A survey of the year 2006 literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast and robust method to determine the energetics of association reactions in solution. The changes in enthalpy, entropy and heat capacity that accompany binding provide unique insights into the balance of forces driving association of molecular entities. ITC is used nowadays on a day-to-day basis in hundreds of laboratories. The method aids projects both in basic and practice-oriented research ranging from medicine and biochemistry to physical chemistry and material sciences. Not surprisingly, the range of studies utilizing ITC data is steadily expanding. In this review, we discuss selected results and ideas that have accumulated in the course of the year 2006, the focus being on biologically relevant systems. Theoretical developments, novel applications and studies that provide a deeper level of understanding of the energetic principles of biological function are primarily considered. Following the appearance of a new generation of titration calorimeters, recent papers provide instructive examples of the synergy between energetic and structural approaches in biomedical and biotechnological research