Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering

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Small-angle X-ray scattering (SAXS) is changing how we perceive biological structures, because it reveals dynamic macromolecular conformations and assemblies in solution. SAXS information captures thermodynamic ensembles, enhances static structures detailed by high-resolution methods, uncovers commonalities among diverse macromolecules, and helps define biological mechanisms. SAXS-based experiments on RNA riboswitches and ribozymes and on DNA–protein complexes including DNA–PK and p53 discover flexibilities that better define structure–function relationships. Furthermore, SAXS results suggest conformational variation is a general functional feature of macromolecules. Thus, accurate structural analyses will require a comprehensive approach that assesses both flexibility, as seen by SAXS, and detail, as determined by X-ray crystallography and NMR. Here, we review recent SAXS computational tools, technologies, and applications to nucleic acids and related structures.

Introduction

Dynamic macromolecular assemblies composed of nucleic acids and proteins integrate the environmental and cellular signals into biological actions. Yet as of 2008, only 5% of the total structures deposited in the PDB were of nucleic or protein–nucleic acid composition, and most macromolecules function in complexes with an average of 5 partners per protein. Macromolecular X-ray crystallography (MX), Nuclear Magnetic Resonance (NMR), and electron microscopy (EM) are our most reliable structural tools; nonetheless, these techniques have limitations for macromolecules with functional flexibility and intrinsic disorder, which occurs in functional regions and interfaces [1, 2]. Therefore, as structural biology evolves, it will need to provide structural insights into larger macromolecular assemblies that display dynamics, flexibility, and disorder.

Section snippets

Solution structures from X-ray scattering

For noncoding functional RNAs and intrinsically disordered proteins, defining their shapes and conformational space in solution marks a critical step toward understanding their functional roles. Facilitating this goal are ab initio bead-modeling algorithms for interpreting small-angle X-ray scattering (SAXS) data. These ab initio models represent low-resolution shapes and contribute significantly to interpretation of flexible systems in solution, particularly for protein–DNA complexes. SAXS is

Ins and outs of SAXS (basic theory)

Under sufficiently dilute conditions, the SAXS profile, I(q), represents the simultaneous scattering measurement of the macromolecule in all orientations. This inherently reduces the resolution, whereas in MX the scattering from the ordered macromolecules produces diffraction intensities that are subsequently transformed to an electron density map. In SAXS, the transformation of the scattering data, I(q) yields the P(r)-distribution, a histogram of the interatomic vectors within the

Bridging the solution divide

A direct application of SAXS is to test the validity of X-ray crystal structures for functionally important macromolecular conformations and assemblies in solution. Crystal packing forces and subsequent cryogenic temperatures needed for MX experiments constrain the structural ensemble and beg the question as to how well the crystal structure models the solution conformation including possible multiple conformational states. Similarly, a crystal structure may be obtained only with a bound

Detecting conformational switching as a control of biological function

The T. maritima lysine-responsive riboswitch, a 161-nucleotide regulatory RNA element found in the 5′UTR of several lysine biosynthetic genes, binds the amino acid lysine and regulates gene expression. Comparison of the crystal structures with the SAXS data of the bound and free states of the riboswitch demonstrated that the riboswitch does not adopt a different conformation in any state suggesting that the functional mechanism of transcriptional regulation does not occur through large-scale

Architectural flexibility and rigidity in biological outcomes

SAXS can be readily combined with biochemical and mutational information to define flexible complexes, despite limited resolution. In MX, flexible regions may contribute to an incomplete structural model because of missing electron density. In SAXS, flexible regions will contribute to the observed intensity giving a complete assessment of the entire macromolecule. Consequently, SAXS measurements from a macromolecule with an incomplete high-resolution structure provide an opportunity to extend

Envisioning whole macromolecular systems

SAXS provides powerful restraints for modeling large macromolecular assemblies [20]. Particularly for folded RNA whose building block generally consists of 22 Å helical measure, a SAXS-based ab initio model can serve as a suitable structural framework for model building. The VS ribozyme is the largest known nucleolytic ribozyme for which there is no determined crystal structure. Decades of research have dissected the ribozyme into its component catalytic pieces, identified catalytically

The conformational ensemble

The SAXS profile is a direct interrogation of the thermodynamic ensemble. Typical synchrotron-based SAXS measurements will measure 10 000 billion molecules simultaneously and for a protein or complex that may contain an intrinsically flexible linker between multiple domains, a single model would be grossly insufficient to explain the data. Provided an initial starting model, two promising approaches for modeling the ensemble are pushing SAXS into an exciting new direction, the Ensemble

Conclusions and outlook

As structural biology advances to include larger and more flexible systems, SAXS is emerging as an all-purpose tool in the structural biologists toolkit. SAXS can characterize sample quality, provide the first structural insights into a novel complex, identify assembly states in solution, and detect functional conformational changes in the presence of substrates or ligands [3, 5••]. Synchrotron-based facilities now extend SAXS into the high-throughput regime where 15 μL samples at 1 mg/mL and

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest.

Acknowledgements

We sincerely thank Alan Fersht and Martin Blackledge for providing material for Figure 3. We thank Jan Lipfert, SIBYLS staff members G Hura and M Hammel for discussions and RT Batey for SAM-I. The authors acknowledge salary and other support for SAXS technologies at the SIBYLS beamline (BL12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory by United States Department of Energy (DOE) program Integrated Diffraction Analysis Technologies (IDAT) under contract

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