Structural studies of macromolecules in solution using small angle X-ray scattering

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Mrozowich, Tyler
McLennan, Steffane
Overduin, Michael
Pater, Trushar R.
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Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attached to laminin y-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.
Open access article. Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported license (CC BY-NC-NC 3.0) applies
Biochemistry , Extracellular matrix protein , Hybrid solution structure , Iaminin , Multidomain protein , Nidogen , Protein interaction , Protein structure , Structural domain , Small angle X-ray scattering , Three-dimension model
Mrozowich, T., McLennan, S., Overduin, M., & Patel, T. R. (2018). Structural studies of macromolecules in solution using small angle X-ray scattering. Journal of Visual Experiments, (141), Article e58538.