Abstract
Small angle X-ray scattering is an increasingly utilized method for characterizing the shape and structural properties of proteins in solution. The technique is amenable to very large protein complexes and to dynamic particles with different conformational states. It is therefore ideally suited to the analysis of some flagellar motor components. Indeed, we recently used the method to analyze the solution structure of the flagellar motor protein FliG, which when combined with high-resolution snapshots of conformational states from crystal structures, led to insights into conformational transitions that are important in mediating the self-assembly of the bacterial flagellar motor. Here, we describe procedures for X-ray scattering data collection of flagellar motor components, data analysis, and interpretation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stock D, Namba K, Lee LK (2012) Nanorotors and self-assembling macromolecular machines: the torque ring of the bacterial flagellar motor. Curr Opin Biotechnol 23:545–554. doi:10.1016/j.copbio.2012.01.008
Kojima S, Furukawa Y, Matsunami H et al (2008) Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor. J Bacteriol 190:3314–3322. doi:10.1128/JB.01710-07
Roujeinikova A (2008) Crystal structure of the cell wall anchor domain of MotB, a stator component of the bacterial flagellar motor: implications for peptidoglycan recognition. Proc Natl Acad Sci USA 105:10348–10353. doi:10.1073/pnas.0803039105
Samatey FA, Matsunami H, Imada K et al (2004) Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism. Nature 431:1062–1068. doi:10.1038/nature02997
Samatey FA, Imada K, Nagashima S et al (2001) Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature 410:331–337. doi:10.1038/35066504
Baker MAB, Hynson RMG, Ganuelas LA et al (2016) Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor. Nat Struct Mol Biol. doi:10.1038/nsmb.3172
Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95:559–571. doi:10.1002/bip.21638
Svergun DI, Koch MHJ, Timmins PA, May RP (2013) Small angle X-ray and neutron scattering from solutions of biological macromolecules. Oxford University Press, Oxford
David G, Pérez J (2009) Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline. J Appl Cryst 42:892–900. doi:10.1107/S0021889809029288
Hynson RMG, Duff AP, Kirby N et al (2015) Differential ultracentrifugation coupled to small-angle X-ray scattering on macromolecular complexes. J Appl Cryst 48:769–775. doi:10.1107/S1600576715005051
Glatter O, IUCr (1977) A new method for the evaluation of small-angle scattering data. J Appl Cryst 10:415–421. doi: 10.1107/S0021889877013879
Svergun DI, IUCr (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 25:495–503. doi: 10.1107/S0021889892001663
Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76:2879–2886. doi:10.1016/S0006-3495(99)77443-6
Petoukhov MV, Konarev PV, Kikhney AG (2007) ATSAS 2.1-towards automated and web-supported small-angle scattering data analysis. J Appl Crystallogr. 40:S223–S228
Jacques DA, Guss JM, Svergun DI, Trewhella J (2012) Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution. Acta Crystallogr D Biol Crystallogr 68:620–626. doi:10.1107/S0907444912012073
Volkov VV, Svergun DI, IUCr (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Cryst 36:860–864. doi: 10.1107/S0021889803000268
Tria G, Mertens HDT, Kachala M, Svergun DI (2015) Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering. IUCrJ 2:207–217. doi:10.1107/S205225251500202X
Pelikan M, Hura GL, Hammel M (2009) Structure and flexibility within proteins as identified through small angle X-ray scattering. Gen Physiol Biophys 28:174–189
Zhu S, Takao M, Li N et al (2014) Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor. Proc Natl Acad Sci USA 111:13523–13528. doi:10.1073/pnas.1324201111
O’Neill J, Xie M, Hijnen M, Roujeinikova A (2011) Role of the MotB linker in the assembly and activation of the bacterial flagellar motor. Acta Crystallogr D Biol Crystallogr 67:1009–1016. doi:10.1107/S0907444911041102
Konarev PV, Volkov VV, Sokolova AV et al (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Cryst 36:1277–1282. doi:10.1107/S0021889803012779
Acknowledgments
This work was supported by Australian Research Council Discovery Project Grant (DP130102219) and Discovery Early Career Research Award (DE140100262), as well as by the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Lee, L.K. (2017). Structural Analysis of the Flagellar Component Proteins in Solution by Small Angle X-Ray Scattering. In: Minamino, T., Namba, K. (eds) The Bacterial Flagellum. Methods in Molecular Biology, vol 1593. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6927-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6927-2_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6926-5
Online ISBN: 978-1-4939-6927-2
eBook Packages: Springer Protocols