Structure and Intermolecular Interactions between L-Type Straight Flagellar Filaments

Abstract

Bacterial mobility is powered by rotation of helical flagellar filaments driven by rotary motors. Flagellin isolated from the Salmonella Typhimurium SJW1660 strain, which differs by a point mutation from the wild-type strain, assembles into straight filaments in which flagellin monomers are arranged in a left-handed helix. Using small-angle x-ray scattering and osmotic stress methods, we investigated the structure of SJW1660 flagellar filaments as well as the intermolecular forces that govern their assembly into dense hexagonal bundles. The scattering data were fitted to models, which took into account the atomic structure of the flagellin subunits. The analysis revealed the exact helical arrangement and the super-helical twist of the flagellin subunits within the filaments. Under osmotic stress, the filaments formed two-dimensional hexagonal bundles. Monte Carlo simulations and continuum theories were used to analyze the scattering data from hexagonal arrays, revealing how the bundle bulk modulus and the deflection length of filaments in the bundles depend on the applied osmotic stress. Scattering data from aligned flagellar bundles confirmed the theoretically predicated structure-factor scattering peak line shape. Quantitative analysis of the measured equation of state of the bundles revealed the contributions of electrostatic, hydration, and elastic interactions to the intermolecular forces associated with bundling of straight semi-flexible flagellar filaments.

D+: Software for High-Resolution Hierarchical Modeling of Solution X-Ray Scattering from Complex Structures

“…Recently, we have used D+ to analyze solution X-ray scattering data (Eisenberg et al, 2017;Asor et al, 2017;Ginsburg et al, 2017;Chung et al, 2017). In particular, we have analyzed solutions of SJW1660 flagellar filaments (Louzon et al, 2017).…”

“…Methods Solution X-ray scattering measurements were performed using energy of 10 keV at ID02 beamline (headed by T. Narayanan) in ESRF (Grenoble), at P12 EMBL BioSAXS beamline (D. Svergun) in PETRA III (DESY, Hamburg), and at SWING beamline (J. Perez) in Soleil synchrotron (Gif-sur-YVETTE), or in our in-house spectrometer, using energy of 8 keV. Detailed experimental descriptions of these setups were provided elsewhere (David & Pérez, 2009;Blanchet et al, 2015;Van Vaerenbergh et al, 2016;Louzon et al, 2017;Fink et al, 2017b). The intensity frames were normalized to the intensity of the transmitted beam, azimuthally averaged (Hammersley, 2016), and background-subtracted as explained below, in SOM §5, and in our earlier publication .…”

“…Usage examples can be found in our recent publications (Ginsburg et al, 2017;Louzon et al, 2017;Asor et al, 2017). To further validate and demonstrate the use of D+, we measured and analyzed the scattering curve from lysozyme and microtubule (MT) in solution.…”

“…Owing to the lack of phase and 3D information (resulting from the free rotation of molecules in solution) data analysis is challenging. Challenges include addressing the contribution to the scattering signal from individual objects and from their arrangement in space, accounting for the contribution of the solvating shell, thermal fluctuations, and involve computational and model fitting aspect (see for example (Louzon et al, 2017)). Multiple computer programs and algorithms have been developed over the past years for analyzing the scattering data from soluble macromolecules or oligomers (Virtanen et al, 2011;Petoukhov & Svergun, 2005; Bardhan et al, 2009;Koutsioubas & Perez, 2013;Schneidman-Duhovny et al, 2010;Schneidman-Duhovny et al, 2013;Poitevin et al, 2011;Knight & Hub, 2015;Franke et al, 2015;Hura et al, 2009;Franke & Svergun, 2009;Spinozzi et al, 2014;Sarje et al, 2014;Grant, 2018;Curtis et al, 2012;Wright & Perkins, 2015;Gumerov et al, 2012;Watson & Curtis, 2013;Ravikumar et al, 2013;Petoukhov et al, 2012;Schneidman-Duhovny et al, 2016;.…”

“…Advanced features including thermal fluctuations and intermolecular interactions can also be taken into account by using the python wrapper of D+ or by writing Lua scripts inside D+, which can extend, modify, or control the arrangements of subunits. The python API of D+ can also be used to integrate D+ with advanced computations or simulations, as demonstrated (Louzon et al, 2017)).…”