Integrated model of the vertebrate augmin complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin\u27s structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation

Accurate Backbone 13C and 15N Chemical Shift Tensors in Galectin-3 Determined by MAS NMR and QM/MM : Details of Structure and Environment Matter

Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate 13C and 15N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for 15N atoms. Here we compare experimentally determined backbone 13Cα and 15NH chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed 15NH chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR

Chemical Shifts of the Carbohydrate Binding Domain of Galectin-3 from Magic Angle Spinning NMR and Hybrid Quantum Mechanics/Molecular Mechanics Calculations

Magic angle spinning NMR spectroscopy is uniquely suited to probe the structure and dynamics of insoluble proteins and protein assemblies at atomic resolution, with NMR chemical shifts containing rich information about biomolecular structure. Access to this information, however, is problematic, since accurate quantum mechanical calculation of chemical shifts in proteins remains challenging, particularly for 15NH. Here we report on isotropic chemical shift predictions for the carbohydrate recognition domain of microcrystalline galectin-3, obtained from using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, implemented using an automated fragmentation approach, and using very high resolution (0.86 Å lactose-bound and 1.25 Å apo form) X-ray crystal structures. The resolution of the X-ray crystal structure used as an input into the AF-NMR program did not affect the accuracy of the chemical shift calculations to any significant extent. Excellent agreement between experimental and computed shifts is obtained for 13Cα, while larger scatter is observed for 15NH chemical shifts, which are influenced to a greater extent by electrostatic interactions, hydrogen bonding, and solvation

Investigating the Formation of a Repulsive Hydrogel of a Cationic 16mer Peptide at Low Ionic Strength in Water by Vibrational Spectroscopy and Rheology

The cationic peptide (AAKA)₄ (AK16) exhibits a high propensity for aggregation into β-sheet-like structures in spite of the high positive charge of its protonated lysine side chains. Upon incubation into an aqueous solution, the peptide maintains a metastable β-sheet-like structure with fibrillar content, the apparent stability of which increases with peptide concentration. In the presence of a sufficiently high concentration of anions, the peptide spontaneously forms a hydrogel at millimolar concentrations. Interestingly, we find that even in the absence of gel-supporting anions, the peptide is capable of forming a hydrogel in the centimolar range. Rheological data reveal that the gel is a stable elastic solid. These data show that the peptide can overcome the repulsive interactions between the positively charged ammonium groups of the lysine residues. The addition of 1 M NaCl just accelerates this process. Atomic force microscopy images of the peptide gel reveal fibrils with thicknesses between 4 and 8 nm, which suggests that they contain multiple layers of sheets. We propose that long tapes of β-sheet are arranged in fibrils via stacking of alternating interfaces induced by hydrophobic interactions between alanine side chains and by the formation of a hydrogen bonded water network between hydrophilic sides of AK16 β-sheets, which leads to the observed immobilization of the solvent in the formed hydrogel. Water immobilization is proposed as the likely cause for a significant increase in the amide I′ oscillator strength of the formed β-sheet structures

¹⁹F Magic Angle Spinning NMR Spectroscopy and Density Functional Theory Calculations of Fluorosubstituted Tryptophans: Integrating Experiment and Theory for Accurate Determination of Chemical Shift Tensors

The ¹⁹F chemical shift is a sensitive NMR probe of structure and electronic environment in organic and biological molecules. In this report, we examine chemical shift parameters of 4F-, 5F-, 6F-, and 7F-substituted crystalline tryptophan by magic angle spinning (MAS) solid-state NMR spectroscopy and density functional theory. Significant narrowing of the ¹⁹F lines was observed under fast MAS conditions, at spinning frequencies above 50 kHz. The parameters characterizing the ¹⁹F chemical shift tensor are sensitive to the position of the fluorine in the aromatic ring and, to a lesser extent, the chirality of the molecule. Accurate calculations of ¹⁹F magnetic shielding tensors require the PBE0 functional with a 50% admixture of a Hartree–Fock exchange term, as well as taking account of the local crystal symmetry. The methodology developed will be beneficial for ¹⁹F-based MAS NMR structural analysis of proteins and protein assemblies

EXCESS workshop: Descriptions of rising low-energy spectra

International audienceMany low-threshold experiments observe sharply rising event rates of yet unknown origins below a few hundred eV, and larger than expected from known backgrounds. Due to the significant impact of this excess on the dark matter or neutrino sensitivity of these experiments, a collective effort has been started to share the knowledge about the individual observations. For this, the EXCESS Workshop was initiated. In its first iteration in June 2021, ten rare event search collaborations contributed to this initiative via talks and discussions. The contributing collaborations were CONNIE, CRESST, DAMIC, EDELWEISS, MINER, NEWS-G, NUCLEUS, RICOCHET, SENSEI and SuperCDMS. They presented data about their observed energy spectra and known backgrounds together with details about the respective measurements. In this paper, we summarize the presented information and give a comprehensive overview of the similarities and differences between the distinct measurements. The provided data is furthermore publicly available on the workshop’s data repository together with a plotting tool for visualization