X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution

Abstract

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 A to 10 A resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein-nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.

Solution structure of RNase P RNA

“…Fitting structure to solution envelope with EOM and NMA A number of computational techniques have emerged in recent years that allow improvement of atomic models to achieve a better agreement with observed SAXS data such as the ensemble optimization method (EOM) (Bernado et al 2007;Putnam et al 2007;Rambo and Tainer 2010a). In EOM, a large set of conformationally different models are generated from a set of starting models to generate a large pool of reasonable conformational alternatives; small ensembles of conformers are then selected from the pool by a Monte Carlo-based genetic algorithm to maximize the agreement between theoretical scattering by a selected ensemble and the experimental data.…”

Asymmetric perturbations of signalling oligomers

“…SAXS provides information on molecular shape, conformation, assembly state, aggregation, flexibility, and can be used predominantly as a complementary tool to high resolution techniques and single-molecule studies (Petoukhov and Svergun, 2007;Rambo and Tainer, 2013). For instance, SAXS in solution can verify physiologically relevant interfaces indicated by XRC, aid the evaluation of weak assemblies observed in crystals, and distinguish between alternative assemblies in different XRC structures (Putnam et al, 2007). Additionally, when high-resolution structures of individual subunits are available, it can be used to determine the relative orientation and position of subunits (Wall et al, 2000).…”

Preparation and Characterization of Bacterial Protein Complexes for Structural Analysis

“…In recent years there has been a revival in the use of SAXS to analyze protein shapes and conformations in solution (Koch et al, 2003;Putnam et al, 2007). The basic experimental design consists of exposing a protein solution to a collimated beam of hard X-rays and detecting X-rays scattered at a small angle.…”

“…Another useful diagnostic tool is the Kratky plot (q vs. I*q 2 ), which is an indicator of the folded state of a protein (Putnam et al, 2007). Folded globular proteins show a typical bell curve whose q value at the maximum can be used to determine an approximate molecular weight, whereas unfolded proteins show a plateau-shaped curve with no distinct peak.…”