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Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering.

ABSTRACT: Dynamic ensembles of macromolecules mediate essential processes in biology. Understanding the mechanisms driving the function and molecular interactions of 'unstructured' and flexible molecules requires alternative approaches to those traditionally employed in structural biology. Small-angle X-ray scattering (SAXS) is an established method for structural characterization of biological macromolecules in solution, and is directly applicable to the study of flexible systems such as intrinsically disordered proteins and multi-domain proteins with unstructured regions. The Ensemble Optimization Method (EOM) [Bernadó et al. (2007 ?). J. Am. Chem. Soc. 129, 5656-5664] was the first approach introducing the concept of ensemble fitting of the SAXS data from flexible systems. In this approach, a large pool of macromolecules covering the available conformational space is generated and a sub-ensemble of conformers coexisting in solution is selected guided by the fit to the experimental SAXS data. This paper presents a series of new developments and advancements to the method, including significantly enhanced functionality and also quantitative metrics for the characterization of the results. Building on the original concept of ensemble optimization, the algorithms for pool generation have been redesigned to allow for the construction of partially or completely symmetric oligomeric models, and the selection procedure was improved to refine the size of the ensemble. Quantitative measures of the flexibility of the system studied, based on the characteristic integral parameters of the selected ensemble, are introduced. These improvements are implemented in the new EOM version 2.0, and the capabilities as well as inherent limitations of the ensemble approach in SAXS, and of EOM 2.0 in particular, are discussed.

SUBMITTER: Tria G

PROVIDER: S-EPMC4392415 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Project description:X-ray scattering of biological macromolecules in solution is an increasingly popular tool for structural biology and benefits greatly from modern high-brightness synchrotron sources. The upgraded MacCHESS BioSAXS station is now located at the 49-pole wiggler beamline G1. The 20-fold improved flux over the previous beamline F2 provides higher sample throughput and autonomous X-ray scattering data collection using a unique SAXS/WAXS dual detectors configuration. This setup achieves a combined q-range from 0.007 to 0.7 Å(-1), enabling better characterization of smaller molecules, while opening opportunities for emerging wide-angle scattering methods. In addition, a facility upgrade of the positron storage ring to continuous top-up mode has improved beam stability and eliminated beam drift over the course of typical BioSAXS experiments. Single exposure times have been reduced to 2 s for 3.560 mg ml(-1) lysozyme with an average quality factor I/σ of 20 in the Guinier region. A novel disposable plastic sample cell design that incorporates lower background X-ray window material provides users with a more pristine sample environment than previously available. Systematic comparisons of common X-ray window materials bonded to the cell have also been extended to the wide-angle regime, offering new insight into best choices for various q-space ranges. In addition, a quantitative assessment of signal-to-noise levels has been performed on the station to allow users to estimate necessary exposure times for obtaining usable signals in the Guinier regime. Users also have access to a new BioSAXS sample preparation laboratory which houses essential wet-chemistry equipment and biophysical instrumentation. User experiments at the upgraded BioSAXS station have been on-going since commissioning of the beamline in Summer 2013. A planned upgrade of the G1 insertion device to an undulator for the Winter 2014 cycle is expected to further improve flux by an order of magnitude.

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Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering.

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