Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths.
ABSTRACT: Obtaining phase information remains a formidable challenge for nucleic acid structure determination. The introduction of an X-ray synchrotron beamline designed to be tunable to long wavelengths at Diamond Light Source has opened the possibility to native de novo structure determinations by the use of intrinsic scattering elements. This provides opportunities to overcome the limitations of introducing modifying nucleotides, often required to derive phasing information. In this paper, we build on established methods to generate new tools for nucleic acid structure determinations. We report on the use of (i) native intrinsic potassium single-wavelength anomalous dispersion methods (K-SAD), (ii) use of anomalous scattering elements integral to the crystallization buffer (extrinsic cobalt and intrinsic potassium ions), (iii) extrinsic bromine and intrinsic phosphorus SAD to solve complex nucleic acid structures. Using the reported methods we solved the structures of (i) Pseudorabies virus (PRV) RNA G-quadruplex and ligand complex, (ii) PRV DNA G-quadruplex, and (iii) an i-motif of human telomeric sequence. Our results highlight the utility of using intrinsic scattering as a pathway to solve and determine non-canonical nucleic acid motifs and reveal the variability of topology, influence of ligand binding, and glycosidic angle rearrangements seen between RNA and DNA G-quadruplexes of the same sequence.
Project description:X-ray diffraction patterns from crystals of biological macromolecules contain sufficient information to define atomic structures, but atomic positions are inextricable without having electron-density images. Diffraction measurements provide amplitudes, but the computation of electron density also requires phases for the diffracted waves. The resonance phenomenon known as anomalous scattering offers a powerful solution to this phase problem. Exploiting scattering resonances from diverse elements, the methods of MAD (multiwavelength anomalous diffraction) and SAD (single-wavelength anomalous diffraction) now predominate for de novo determinations of atomic-level biological structures. This review describes the physical underpinnings of anomalous diffraction methods, the evolution of these methods to their current maturity, the elements, procedures and instrumentation used for effective implementation, and the realm of applications.
Project description:De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements - primarily sulfur in proteins and phospho-rus in nucleic acids - increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10?µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5?keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1?200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.
Project description:Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Zmax ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f'' scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.
Project description:Phaser is a program that implements likelihood-based methods to solve macromolecular crystal structures, currently by molecular replacement or single-wavelength anomalous diffraction (SAD). SAD phasing is based on a likelihood target derived from the joint probability distribution of observed and calculated pairs of Friedel-related structure factors. This target combines information from the total structure factor (primarily non-anomalous scattering) and the difference between the Friedel mates (anomalous scattering). Phasing starts from a substructure, which is usually but not necessarily a set of anomalous scatterers. The substructure can also be a protein model, such as one obtained by molecular replacement. Additional atoms are found using a log-likelihood gradient map, which shows the sites where the addition of scattering from a particular atom type would improve the likelihood score. An automated completion algorithm adds new sites, choosing optionally among different atom types, adds anisotropic B-factor parameters if appropriate and deletes atoms that refine to low occupancy. Log-likelihood gradient maps can also identify which atoms in a refined protein structure are anomalous scatterers, such as metal or halide ions. These maps are more sensitive than conventional model-phased anomalous difference Fouriers and the iterative completion algorithm is able to find a significantly larger number of convincing sites.
Project description:Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here, we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing the signal-to-noise ratio in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multicrystal SAD method (5 to 13 equivalent crystals), we determined structures at modest resolution (2.8 to 2.3 angstroms) for native proteins varying in size (127 to 1148 unique residues) and number of sulfur sites (3 to 28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.
Project description:Native single-wavelength anomalous dispersion (SAD) is the most attractive de novo phasing method in macromolecular crystallography, as it directly utilizes intrinsic anomalous scattering from native crystals. However, the success of such an experiment depends on accurate measurements of the reflection intensities and therefore on careful data-collection protocols. Here, the low-dose, multiple-orientation data-collection protocol for native SAD phasing developed at beamline X06DA (PXIII) at the Swiss Light Source is reviewed, and its usage over the last four years on conventional crystals (>50?µm) is reported. Being experimentally very simple and fast, this method has gained popularity and has delivered 45 de novo structures to date (13 of which have been published). Native SAD is currently the primary choice for experimental phasing among X06DA users. The method can address challenging cases: here, native SAD phasing performed on a streptavidin-biotin crystal with P21 symmetry and a low Bijvoet ratio of 0.6% is highlighted. The use of intrinsic anomalous signals as sequence markers for model building and the assignment of ions is also briefly described.
Project description:Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9?Å is demonstrated for native-SAD phasing on a 266?kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86?kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3?Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3?Å are discussed.
Project description:A fast Fourier transform (FFT) method is described for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by X-ray single-wavelength anomalous diffraction (SAD). This method is based on the maximum-likelihood SAD phasing function, which accounts for measurement errors and for correlations between the observed and calculated Bijvoet mates. Proof of principle is shown that this method can improve determination of the anomalously scattering substructure in challenging cases where the anomalous scattering from the substructure is weak but the substructure also constitutes a significant fraction of the real scattering. The method is deterministic and can be fast compared with existing multi-trial dual-space methods for SAD substructure determination.
Project description:We describe a likelihood-based method for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. With the use of partial models and electron density maps in searches for anomalously scattering atoms, testing of alternative values of parameters and parallelized automated model-building, this method has the potential to extend the applicability of the SAD method in challenging cases.
Project description:Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable.