Macromolecular X-ray Crystallography Research Articles

Optimizing the spatial distribution of dose in X-ray macromolecular crystallography

X-ray data collection for macromolecular crystallography can lead to highly inhomogeneous distributions of dose within the crystal volume for cases when the crystal is larger than the beam or when the beam is non-uniform (gaussian-like), particularly when crystal rotation is fully taken into account. Here the spatial distribution of dose is quantitatively modelled in order to compare the effectiveness of two dose-spreading data-collection protocols: helical scanning and translational collection. Their effectiveness in reducing the peak dose per unit diffraction is investigated via simulations for four common crystal shapes (cube, plate, long and short needles) and beams with a wide range of full width half maximum values. By inspection of the chosen metric, it is concluded that the optimum strategy is always to use as flat (top-hat) a beam as possible and to either match the beam size in both dimensions to the crystal, or to perform a helical scan with a beam which is narrow along the rotation axis and matched to the crystal size along the perpendicular axis. For crystal shapes where this is not possible, the reduction in peak dose per unit diffraction achieved through dose spreading is quantified and tabulated as a reference for experimenters.

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group1-3. JoVE video articles describing the method are available1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group1-3. JoVE video articles describing the method are available1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.

X-ray diffraction as a tool in the path from the design of an active pharmaceutical ingredient to the tablet on the shelf

Accurate knowledge of molecular structure is a prerequisite for rational drug design. X-ray diffraction has a paramount importance in providing the required structural information: the structural determination by X-ray diffraction of new drug candidates and intermediates can provide valuable information of new syntheses and parameters for quantitative structure–activity relationships. Macromolecular X-ray crystallography is an important and powerful technique used in the discovery of new medicines. The detailed analysis of crystal structures of protein–ligand complexes allows the study of the specific interactions of a particular drug with its protein target at the atomic level. Most marketed pharmaceuticals consist of molecular crystals. The arrangement of the molecules in a crystal determines its physical properties and greatly influences the processing and formulation of solid pharmaceuticals, as well as key drug properties such as dissolution rate and stability. X-ray diffraction is the main tool to characterize the drugs in solid forms. An example of the use of X-ray diffraction in the development of rifamycins is discussed.

Reduction of radiation damage and other benefits of short wavelengths for macromolecular crystallography data collection

Circumventing radiation damage remains a major problem for X-ray macromolecular crystallography. Analysis of diffraction data collected from normal-sized cryocooled lysozyme crystals shows that the dose required to collect a data set of prescribed resolution and signal-to-noise ratio, assuming an ideally efficient detector, decreases with increasing photon energy in the investigated 6.5–33 keV range. For example, the data collection efficiency is increased by a factor of ∼8 from 8 to 33 keV. Monte Carlo simulations on lysozyme crystals in the range 5–80 keV, taking into account electron escape from samples of different size, also show a positive effect of high energy (albeit less pronounced than in experiments), especially for micrometre-sized samples, and suggest that the optimum energy range is ∼24–41 keV, depending on crystal size. The importance of counting pixel detectors with a good efficiency at high energy is underlined. Macromolecular crystallography beamlines should be modified, or purposely designed, in order to benefit from higher-energy radiation through reduction of global radiation damage, better data accuracy and extension of phasing by anomalous dispersion.

The prominent role of resonant elastic scattering for solving the X-ray structure of macromolecules

This article is a short overview of basic principles, methods and instrumentation of phasing methods based on anomalous scattering for X-ray macromolecular crystallography (MX).

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses 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. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.

Difficult Macromolecular Structures Determined Using X-ray Diffraction Techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.

Highlights from the Macromolecular X-ray Crystallography Facility at Pennsylvania State University (a) crystal structures of bacterial expansin in complex with cellohexoase and β-glucan (b) solution scattering and crystal structure of sorbitol dehydrogenase with a substrate (c) computation model of human Ron receptor with its juxta-membrane domain and (d) model of phospholipase a2 with procyanidins

Highlights from the Macromolecular X-ray Crystallography Facility at Pennsylvania State University (<i>a</i>) crystal structures of bacterial expansin in complex with cellohexoase and β-glucan (<i>b</i>) solution scattering and crystal structure of sorbitol dehydrogenase with a substrate (<i>c</i>) computation model of human Ron receptor with its juxta-membrane domain and (<i>d</i>) model of phospholipase a2 with procyanidins

Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate.

Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50-250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e(-)Å(-2) s(-1) or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments.

Similarities and differences in radiation damage at 100 Kversus160 K in a crystal of thermolysin

The temperature-dependence of radiation damage in macromolecular X-ray crystallography is currently much debated. Most protein crystallographic studies are based on data collected at 100 K. Data collection at temperatures below 100 K has been proposed to reduce radiation damage and above 100 K to be useful for kinetic crystallography that is aimed at the generation and trapping of protein intermediate states. Here the global and specific synchrotron-radiation sensitivity of crystalline thermolysin at 100 and 160 K are compared. Both types of damage are higher at 160 K than at 100 K. At 160 K more residue types are affected (Lys, Asp, Gln, Pro, Thr, Met, Asn) than at 100 K (Met, Asp, Glu, Lys). The X-ray-induced relative atomic B-factor increase is shown to correlate with the proximity of the atom to the nearest solvent channel at 160 K. Two models may explain the observed correlation: either an increase in static disorder or an increased attack of hydroxyl radicals from the solvent area of the crystal.

Polarizable Atomic Multipole X-Ray Refinement: Particle Mesh Ewald Electrostatics for Macromolecular Crystals.

Refinement of macromolecular models from X-ray crystallography experiments benefits from prior chemical knowledge at all resolutions. As the quality of the prior chemical knowledge from quantum or classical molecular physics improves, in principle so will resulting structural models. Due to limitations in computer performance and electrostatic algorithms, commonly used macromolecules X-ray crystallography refinement protocols have had limited support for rigorous molecular physics in the past. For example, electrostatics is often neglected in favor of nonbonded interactions based on a purely repulsive van der Waals potential. In this work we present advanced algorithms for desktop workstations that open the door to X-ray refinement of even the most challenging macromolecular data sets using state-of-the-art classical molecular physics. First we describe theory for particle mesh Ewald (PME) summation that consistently handles the symmetry of all 230 space groups, replicates of the unit cell such that the minimum image convention can be used with a real space cutoff of any size and the combination of space group symmetry with replicates. An implementation of symmetry accelerated PME for the polarizable atomic multipole optimized energetics for biomolecular applications (AMOEBA) force field is presented. Relative to a single CPU core performing calculations on a P1 unit cell, our AMOEBA engine called Force Field X (FFX) accelerates energy evaluations by more than a factor of 24 on an 8-core workstation with a Tesla GPU coprocessor for 30 structures that contain 240 000 atoms on average in the unit cell. The benefit of AMOEBA electrostatics evaluated with PME for macromolecular X-ray crystallography refinement is demonstrated via rerefinement of 10 crystallographic data sets that range in resolution from 1.7 to 4.5 Å. Beginning from structures obtained by local optimization without electrostatics, further optimization using AMOEBA with PME electrostatics improved agreement of the model with the data (Rfree was lowered by 0.5%), improved geometric features such as favorable (ϕ, ψ) backbone conformations, and lowered the average potential energy per residue by over 10 kcal/mol. Furthermore, the MolProbity structure validation tool indicates that the geometry of these rerefined structures is consistent with X-ray crystallographic data collected up to 2.2 Å, which is 0.9 Å better than the actual mean quality (3.1 Å). We conclude that polarizable AMOEBA-assisted X-ray refinement offers advantages to methods that neglect electrostatics and is now efficient enough for routine use.

Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide.

Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation.

High-Resolution X-ray Crystallography of Bovine H-Protein at 0.88 Å Resolution

Recent technical developments in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, numbers of high-resolution structures are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 A resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium- and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics and R factors against the final model and the number of visualized hydrogen atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of hydrogen atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data.

A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results

Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined.

High-resolution X-ray crystal structure of bovine H-protein at 0.88 Å resolution

Recent technical improvements in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, examples of high-resolution structure determination are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 A resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium- and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics and R factors against the final model and the number of visualized H atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of H atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data.

PHENIX: a comprehensive Python-based system for macromolecular structure solution

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

Synthesis of a 4‐Selenothymidine Phosphoramidite and Incorporation into Oligonucleotides

The detailed synthetic protocol for a 4-selenothymidine phosphoramidite and its use to prepare modified oligonucleotides is described here. The Se-phosphoramidite synthesis was achieved by developing a useful protection and deprotection system for the selenium functionality. The coupling reaction of the Se-phosphoramidite during solid-phase oligonucleotide synthesis is quantitative, and the oligonucleotides containing the Se-modification are stable. Based on crystal structure analysis, the selenium-modified oligonucleotides retain base-pairing like their native counterparts, and the derivatized DNA structure is virtually identical to the native structure. This achievement will present a novel opportunity for structural studies of nucleic acids and their protein complexes, because selenium can resolve the phase problem in macromolecular X-ray crystallography. In addition, this atom-specific replacement of oxygen with selenium will provide a useful tool for investigating biochemical and biophysical properties of nucleic acids and their protein complexes.