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The breathing motions of proteins are thought to play a critical role in function. However, current techniques to study key collective motions are limited to spectroscopy and computation. We present a high-resolution experimental approach based on the total scattering from protein crystals at room temperature (TS/RT-MX) that captures both structure and collective motions. To reveal the scattering signal from protein motions, we present a general workflow that enables robust subtraction of lattice disorder. The workflow introduces two methods: GOODVIBES, a detailed and refinable lattice disorder model based on the rigid-body vibrations of a crystalline elastic network; and DISCOBALL, an independent method of validation that estimates the displacement covariance between proteins in the lattice in real space. Here, we demonstrate the robustness of this workflow and further demonstrate how it can be interfaced with MD simulations towards obtaining high-resolution insight into functionally important protein motions.

The naturally occurring R68S substitution of phenylalanine hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of phenylalanine, in contrast to the WT enzyme. Analytical ultracentrifugation establishes that the isolated regulatory domain of R68S PheH is predominantly monomeric in the absence of phenylalanine and dimerizes in its presence, similar to the regulatory domain of the WT enzyme. Fluorescence and small-angle X-ray scattering analyses establish that the overall conformation of the resting form of R68S PheH is different from that of the WT enzyme. The data are consistent with the substitution disrupting the interface between the catalytic and regulatory domains of the enzyme, shifting the equilibrium between the resting and activated forms ∼200-fold, so that the resting form of R68S PheH is ∼70% in the activated conformation. However, R68S PheH loses activity 2 orders of magnitude more rapidly than the WT enzyme at 37 °C and is significantly more sensitive to proteolysis. We propose that, even though this substitution converts the enzyme to a constitutively active enzyme, it results in PKU because of the decrease in protein stability.

Correlated motions in proteins arising from the collective movements of residues have long been proposed to be fundamentally important to key properties of proteins, from allostery and catalysis to evolvability. Recent breakthroughs in structural biology have made it possible to capture proteins undergoing complex conformational changes, yet intrinsic correlated motions within a conformation remain one of the least understood facets of protein structure. For many decades, the analysis of total X-ray scattering held the promise of animating crystal structures with correlated motions. With recent advances in both X-ray detectors and data interpretation methods, this long-held promise can now be met. In this Perspective, we will introduce how correlated motions are captured in total scattering and provide guidelines for the collection, interpretation, and validation of data. As structural biology continues to push the boundaries, we see an opportunity to gain atomistic insight into correlated motions using total scattering as a bridge between theory and experiment.

A method is introduced to deconvolve SAXS datasets from evolving mixtures, such as those produced by time-resolved, temperature jump, ligand titration and chromatography-coupled setups. The method incorporates general restraints using regularized alternating least squares (REGALS), enabling physically meaningful deconvolution without the need for a hard physicochemical or structural model., Mixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori. To address this issue, the REGALS method (regularized alternating least squares) is introduced, which incorporates simple expectations about the data as prior knowledge, and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, making it well suited for exploring datasets with unknown species. Here, REGALS is applied to the analysis of experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing and time-resolved temperature jump. Based on its performance with these challenging datasets, it is anticipated that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and Python and is available freely as an open-source software package.

Significance Negative feedback regulation of ribonucleotide reductase (RNR) activity by dATP is important for maintaining balanced intracellular 2ʹ-deoxynucleoside triphosphate (dNTP) pools essential for the high fidelity of DNA replication and repair. To date, this type of allostery has been nearly universally associated with dATP binding to the N-terminal ATP-cone domain of the class Ia RNR large subunit (canonical α2), resulting in an altered quaternary structure that is unable to productively bind the second subunit (β2). Here, we report our studies on activity inhibition by dATP of the Bacillus subtilis class Ib RNR, which lacks a traditional ATP-cone domain. This unprecedented allostery involves deoxyadenosine 5′-monophosphate (dAMP) binding to a newly identified site in a partial N-terminal cone domain, forming an unprecedented noncanonical α2., The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme’s quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5′-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.

Over the past century, X-ray crystallography has been defined by a pursuit for perfection and high resolution. In next Holy Grail of crystallography is to embrace imperfection towards a dynamic picture of enzymes.

Protein dynamics are integral to biological function, yet few techniques are sensitive to collective atomic motions. A long-standing goal of X-ray crystallography has been to combine structural information from Bragg diffraction with dynamic information contained in the diffuse scattering background. However, the origin of macromolecular diffuse scattering has been poorly understood, limiting its applicability. We present a finely sampled diffuse scattering map from triclinic lysozyme with unprecedented accuracy and detail, clearly resolving both the inter- and intramolecular correlations. These correlations are studied theoretically using both all-atom molecular dynamics and simple vibrational models. Although lattice dynamics reproduce most of the diffuse pattern, protein internal dynamics, which include hinge-bending motions, are needed to explain the short-ranged correlations revealed by Patterson analysis. These insights lay the groundwork for animating crystal structures with biochemically relevant motions., Protein motion in crystals causes diffuse X-ray scattering, which so far has been very challenging to measure and interpret. Here the authors present a finely sampled diffuse scattering map from triclinic lysozyme, which allows them to resolve inter- and intramolecular correlations and they further analyze the maps using all-atom molecular dynamics simulations and simple vibrational models, revealing the contribution of internal protein motion.

Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems.

X-ray scattering is uniquely suited to the study of disordered systems and thus has the potential to provide insight into dynamic processes where diffraction methods fail. In particular, while X-ray crystallography has been a staple of structural biology for more than half a century and will continue to remain so, a major limitation of this technique has been the lack of dynamic information. Solution X-ray scattering has become an invaluable tool in structural and mechanistic studies of biological macromolecules where large conformational changes are involved. Such systems include allosteric enzymes that play key roles in directing metabolic fluxes of biochemical pathways, as well as large, assembly-line type enzymes that synthesize secondary metabolites with pharmaceutical applications. Furthermore, crystallography has the potential to provide information on protein dynamics via the diffuse scattering patterns that are overlaid with Bragg diffraction. Historically, these patterns have been very difficult to interpret, but recent advances in X-ray detection have led to a renewed interest in diffuse scattering analysis as a way to probe correlated motions. Here, we will review X-ray scattering theory and highlight recent advances in scattering-based investigations of protein solutions and crystals, with a particular focus on complex enzymes.

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the pre-activated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

Osmolytes are small, chemically diverse, organic solutes that function as an essential component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, and nonprotecting osmolytes, such as urea, destabilize protein structures. Although much is known about osmolyte effects on proteins, less is understood about osmolyte effects on nucleic acids and their counterion atmospheres. Nonprotecting osmolytes destabilize nucleic acid structures, but effects of protecting osmolytes depend on numerous factors including the type of nucleic acid and the complexity of the functional fold. To begin quantifying protecting osmolyte effects on nucleic acid interactions, we used small-angle X-ray scattering (SAXS) techniques to monitor DNA duplexes in the presence of sucrose. This protecting osmolyte is a commonly used contrast matching agent in SAXS studies of protein-nucleic acid complexes; thus, it is important to characterize interaction changes induced by sucrose. Measurements of interactions between duplexes showed no dependence on the presence of up to 30% sucrose, except under high Mg(2+) conditions where stacking interactions were disfavored. The number of excess ions associated with DNA duplexes, reported by anomalous small-angle X-ray scattering (ASAXS) experiments, was sucrose independent. Although protecting osmolytes can destabilize secondary structures, our results suggest that ion atmospheres of individual duplexes remain unperturbed by sucrose.

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson-Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners.

The fundamental structures of genome packaging in the nucleus are nucleosomes, whose stability and dynamics provide key mechanisms for the epigenetic control of genes. The canonical nucleosome consists of 147 basepairs of DNA tightly wrapped around an octamer of histone proteins. In vitro and in vivo studies reveal a growing diversity of nucleosomal structures with varying DNA conformations and histone compositions that facilitate epigenetic activity. Since characterization of the kinetic pathways between different biologically relevant nucleosomes species remain elusive, we developed a novel biophysical method to probe the coordination between changes in DNA conformation and reconfiguration of the histone core.

DNA packages into compact chromatin structure in eukaryotic cell despite its high negative charge. Nucleosome core particles (NCP) are the fundamental repeating units of chromatin and modulate DNA accessibility for gene expression and regulation. NCPs contain a symmetric histone octamer wrapped by 147-bp DNA. The dynamics of DNA packaging and unpackaging from NCPs affects all DNA-based chemistries, but is not well understood due to a lack of structures of the partially unwrapped, kinetic intermediates. Here we applied a novel strategy combining contrast variation with time-resolved small angle x-ray scattering to determine the structures of protein and DNA constituents of NCPs during salt-induced disassembly. We monitored DNA conformation and protein dissociation of NCPs with two positioning sequences: Widom 601 and 5S DNA. For the Widom 601 construct, we measure a transient structure on the millisecond time scale where the DNA is asymmetrically released from a disrupted histone core, and the proteins remain bound to unwrapped DNA in a semi-open conformation. We hypothesis this conformation may be biologically important substrate for gene regulation. The 5S construct also displays asymmetric DNA release, but exhibits a different pattern of protein dissociation. Our results establish a powerful platform for studying the global dynamics of nucleoprotein complexes.

The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders for cryoSAXS have been microfabricated to address these challenges. They have low background scattering and X-ray absorption, require only 640 nl of sample, and allow reproducible sample cooling. Data collected in the sample holders from a nominal illuminated sample volume of 2.5 nl are reproducible down toq≃ 0.02 Å−1, agree with previous cryoSAXS work and are of sufficient quality for reconstructions that match measured crystal structures. These sample holders thus allow faster, more routine cryoSAXS data collection. Additional development is required to reduce sample fracturing and improve data quality at lowq.

Nucleic acids are highly charged polyelectrolytes that interact strongly with salt ions. Rigid, base-paired regions are successfully described with worm like chain models, but non base-paired single stranded regions have fundamentally different polymer properties because of their greater flexibility. Recently, attention has turned to single stranded nucleic acids due to the growing recognition of their biological importance, as well as the availability of sophisticated experimental techniques sensitive to the conformation of individual molecules. We investigate polyelectrolyte properties of poly(dT), an important and widely studied model system for flexible single stranded nucleic acids, in physiologically important mixed mono- and di-valent salt. We report measurements of the form factor and interparticle interactions using SAXS, end to end distances using smFRET, and number of excess ions using ASAXS. We present a coarse-grained model that accounts for flexibility, excluded volume, and electrostatic interactions in these systems. Predictions of the model are validated against experiment. We also discuss the state of all-atom, explicit solvent Molecular Dynamics simulations of poly(dT), the next step in understanding the complexities of ion interactions with these highly charged and flexible polymers.

Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10–20 μL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.

RNA folds efficiently in the presence of divalent ions, Mg2+ ions in particular. In fact, enhanced charge screening by divalent ions is a recurring theme in the study of nucleic acid electrostatics. Although site-specific ions have been identified in RNA crystal structures, the counterion atmosphere that screens the RNA charge is largely composed of a diffuse and mobile ion cloud. Here, we use Small Angle X-ray Scattering (SAXS), Fluorescence Correlation Spectroscopy (FCS) and microfluidic mixing kinetics to investigate the role of ionic strength and ion valence in the interactions, collapse and folding of small model RNA molecules. Our measurements suggest that in the presence of divalent ions the effective charge density near the RNA surface is smaller. We propose that a tighter localization of divalent ions leads to a larger degree of charge compensation and facilitates RNA folding.

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths ( l p ) of ssRNA. We observe valence and ionic strength-dependent differences in l p in a direct comparison between 40-mers of deoxythymidylate (dT 40 ) and uridylate (rU 40 ) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.

Much attention has been focused on DNA condensation because of its fundamental biological importance. The recent discovery of new roles for RNA duplexes demands efficient packaging of double-stranded RNA for therapeutics. Here we report measurements of short DNA and RNA duplexes in the presence of trivalent ions. Under conditions where UV spectroscopy indicates condensation of DNA duplexes into (insoluble) precipitates, RNA duplexes remain soluble. Small angle x-ray scattering results suggest that the differing surface topologies of RNA and DNA may be crucial in generating the attractive forces that result in precipitation.

DNA condensation is of great interest due to its fundamental biological importance. With the discovery of the important roles of RNAi, recent attention has been focused on efficient packaging of dsRNA for therapeutics. In this study, we applied UV spectroscopic and small angle x-ray scattering to investigate the mechanism of RNA condensation. Our results show that double-strand DNA and RNA behave very differently under certain ionic conditions. The forces that lead to side-by-side attraction and subsequent condensation of DNA molecules may be highly correlated with the differing geometric property of RNA and DNA.

Osmolytes, which function as a vital component of the cellular stress response, are small, chemically diverse, intracellular organic solutes. Protecting osmolytes enhance protein stability via preferential exclusion, where denaturation of the protein in the presence of the osmolyte is less favorable than in an aqueous environment. Thus, the correct ratios of protecting to non-protecting osmolytes and protecting osmolytes to ions are critical to maintain protein structure and protein-nucleic acid interactions. In contrast to the effects of osmolytes on protein stability, structure, and function, there is much less understood concerning the effects of osmolytes on nucleic acids. Although non-protecting osmolytes can destabilize both protein and nucleic acid structures, protecting osmolytes have different effects depending on the complexity of the nucleic acid structure. Furthermore, the influence of osmolytes on the ion atmosphere surrounding nucleic acids is not well understood. As a first step in quantifying the effects of osmolytes on nucleic acid electrostatics we used small angle x-ray scattering (SAXS) techniques to monitor 25-bp DNA duplexes and their interactions in the presence and absence of sucrose, a protecting osmolyte and important contrast matching agent in SAXS studies of protein-nucleic acid complexes. Results will be discussed.

The majority of charge-compensating ions around nucleic acids form a diffuse counterion “cloud” that is not amenable to investigation by traditional methods that rely on rigid structural interactions. Although various techniques have been employed to characterize the ion atmosphere around nucleic acids, only anomalous small-angle X-ray scattering (ASAXS) provides information about the spatial distribution of ions. Here we present an experimentally straightforward extension of ASAXS that can be used to count the number of ions around nucleic acids.

RNA and DNA helices have the same charge, −2e/bp, but different helical structures. The 2′-OH present in RNA hinders duplex flexibility and promotes the A-form helix whereas the more malleable and polymorphic DNA duplexes prefer the B-form. Using a combination of experimental and computational approaches, we show that the topology of the A-form helix alters the spatial distribution of counterions and is essential in promoting the charge screening efficiency of RNA helices. Results from Anomalous Small-Angle X-ray Scattering (ASAXS) experiments suggest that monovalent and divalent cations are more closely localized to the RNA central axis due to A-form major groove penetration. This leads to very efficient change screening in RNA helices which has implications for ion-mediated RNA interactions in two important areas: RNA folding reactions and the design of short RNA helices for RNA interference applications.

A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb(+) and Sr(2+)) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein-Zernike equations, with results from the Kovalenko-Hirata closure being closest to experiment for the cases studied here.

Cells transmit and process genetic information using single-stranded RNA (ssRNA); these molecules realize their function through dynamic structural changes such as folding, association with binding partners, and conformational switching. Disordered, un-basepaired states of RNA play a key role in these dynamic structural transitions, yet most relevant measurements of single-stranded oligonucleotides have focused on DNA. Thus, we measure and compare the conformations of chemically similar ssRNA and ssDNA oligonucleotides lacking secondary structure. SAXS curves and smFRET efficiencies of rU40 and dT40 in 100mM NaCl are well-described by a simple wormlike chain model. We detect subtle but significant differences between the contour and persistence lengths of rU40 and dT40 that agree with predictions based on relative sugar pucker preferences of the two nucleotides. To compare the polyelectrolyte properties of ssRNA and ssDNA, we report persistence lengths derived from smFRET data acquired over a wide range of ionic conditions. We find that the apparent charge screening efficiency of divalent magnesium is anomalously large compared to monovalent sodium in both ssRNA and ssDNA. This strong interaction between divalent ions and disordered RNA presents an important problem for polyelectrolyte theory and challenges our understanding of how ions influence RNA folding.

The number and spatial distribution of small positively-charged ions around highly negatively charged DNA or RNA contribute to the free energy of binding in vitro and in vivo. However, the majority of charge compensating ions around nucleic acids forms a diffuse counterion “cloud” that is not amenable to investigation by traditional methods that rely on rigid structural interactions. With 2 x-ray energies, one near and the other 100 eV away from the ion absorption edge, we have successfully used Anomalous Small-Angle X-ray Scattering (ASAXS) to compare and differentiate the ion spatial distribution around comparably sequenced short DNA and RNA helices. Here, we present further information gained when using multiple x-ray energies (up to 5) in an ASAXS experiment. We describe proper treatment of multiple-energy SAXS data including absolute SAXS intensity calibration and measurement of scattering factors from x-ray fluorescence. We discuss the strengths and limitations of this approach and derive useful parameters in depicting the nucleic acid ion atmosphere.

Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na(+), K(+), Rb(+), or Cs(+) counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.

RNA molecules play critical roles in many cellular processes. Traditionally viewed as genetic messengers, RNA molecules were recently discovered to have diverse functions related to gene regulation and expression. RNA also has great potential as a therapeutic and a tool for further investigation of gene regulation. Metal ions are an integral part of RNA structure and should be considered in any experimental or theoretical study of RNA. Here, we report a multidisciplinary approach that combines anomalous small-angle x-ray scattering and molecular-dynamics (MD) simulations with explicit solvent and ions around RNA. From experiment and simulation results, we find excellent agreement in the number and distribution of excess monovalent and divalent ions around a short RNA duplex. Although similar agreement can be obtained from a continuum description of the solvent and mobile ions (by solving the Poisson-Boltzmann equation and accounting for finite ion size), the use of MD is easily extended to flexible RNA systems with thermal fluctuations. Therefore, we also model a short RNA pseudoknot and find good agreement between the MD results and the experimentally derived solution structures. Surprisingly, both deviate from crystal structure predictions. These favorable comparisons of experiment and simulations encourage work on RNA in all-atom dynamic models.

Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution structures of macromolecules and complexes in solution. However, the susceptibility of biomolecule solutions to damage by ionizing radiation can complicate SAXS experiments. Many potentially interesting proteins, such as light sensors and metalloenzymes, require flow cells to distribute the X-ray dose over a large volume. The high sample consumption in these cases can be prohibitive. To circumvent radiation damage, we explore whether cryo-cooling of samples to temperatures of 100 K can prevent aggregation and fragmentation during data collection. We identify SAXS-friendly cryoprotectant conditions that suppress ice formation upon rapid cooling, and compare cryo-SAXS profiles with room temperature measurements for a variety of standard molecules. From scattering volumes as small as 100 nL, we obtain data of sufficient quality for envelope reconstruction, and find good agreement between cryo-SAXS data and known atomic structures. Strikingly, cryo-cooled samples can withstand doses that are 2-3 orders of magnitude higher than typically used for SAXS at room temperature, comparable to those used in cryo-crystallography. While practical challenges remain, this breakthrough opens the possibility of using SAXS with new, high brightness X-ray sources for high throughput applications.

Using complementary time-resolved biochemical and x-ray probes of RNA structure in solution, we investigate the cation-induced folding of the glmS ribozyme, a metabolite-sensing RNA switch that regulates gene expression in bacteria. Hydroxyl radical footprinting experiments have shown a concerted folding transition within the first 10 seconds after adding magnesium. From small angle x-ray scattering (SAXS) experiments performed under similar conditions, we find that native tertiary contact formation is preceded by the collapse of the molecule to a relatively compact intermediate. The subsequent compaction observed by SAXS correlates temporally with changes in hydroxyl radical protection. We propose a structural model for the intermediate and possible implications for the role of secondary structure and electrostatics in the folding process of this ribozyme.

Unstructured regions of RNA molecules require flexibility to accomplish many biological tasks such as conformational switching and protein recognition. Due to its highly charged backbone, the flexibility of single-stranded RNA is influenced by counterions. In this presentation, we continue to explore RNA flexibility using single-stranded nucleic acid homopolymers as a model system [1]. We investigate the role of counterion valence in nucleic acid flexibility using a combination of small-angle X-ray scattering (SAXS) and single-molecule Forster resonance energy transfer (smFRET). We also study how charge-screening of these model systems are affected by mono- and divalent ions in competition. The results imply that various factors can alter the polymeric properties of unstructured nucleic acids, and may be important for tuning RNA conformational dynamics in vivo.Reference: [1] Chen et al. PNAS 2012 109 (3) 799-804

Small angle x-ray scattering (SAXS) gives structural information about biological molecules in solution. However, large (∼30 microliter) sample volumes are needed to mitigate radiation damage, limiting the use of SAXS in studying rare molecules. By cryocooling SAXS samples, radiation damage and required sample volumes are reduced by orders of magnitude [1], but challenges in creating identically-sized frozen samples complicate background subtraction. Here we present microfabricated silicon sample holders for cryoSAXS. These rigid sample holders have a fixed x-ray path length, simplifying background subtraction. Less than 800 nL of sample are required, facilitating measurements on expensive or hard-to-express molecules. These fixed path length, low volume sample holders make cryoSAXS a more accessible technique capable of probing a wide range of biological molecules.1. S. P. Meisburger et al. Biophys. J. 104, 227 (2013).