Single-wavelength Anomalous Diffraction Phasing Research Articles

Native SAD phasing at room temperature.

Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving macromolecular structures. This technique requires the accurate measurement of intensities to determine differences between Bijvoet pairs. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, a strategy is presented to obtain high-quality data from room-temperature, single-crystal experiments. To illustrate the strengths of this approach, native SAD phasing at 6.55 keV was used to solve four structures of three model systems at 295 K. The resulting data sets allow automatic phasing and model building, and reveal alternate conformations that reflect the structure of proteins at room temperature.

A novel class of Candida glabrata cell wall proteins with β-helix fold mediates adhesion in clinical isolates.

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed β-helix domain that is linked to a C-terminal β-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with β-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

Likelihood-based estimation of substructure content from single-wavelength anomalous diffraction (SAD) intensity data.

SAD phasing can be challenging when the signal-to-noise ratio is low. In such cases, having an accurate estimate of the substructure content can determine whether or not the substructure of anomalous scatterer positions can successfully be determined. Here, a likelihood-based target function is proposed to accurately estimate the strength of the anomalous scattering contribution directly from the measured intensities, determining a complex correlation parameter relating the Bijvoet mates as a function of resolution. This gives a novel measure of the intrinsic anomalous signal. The SAD likelihood target function also accounts for correlated errors in the measurement of intensities from Bijvoet mates, which can arise from the effects of radiation damage. When the anomalous signal is assumed to come primarily from a substructure comprising one anomalous scatterer with a known value of f'' and when the protein composition of the crystal is estimated correctly, the refined complex correlation parameters can be interpreted in terms of the atomic content of the primary anomalous scatterer before the substructure is known. The maximum-likelihood estimation of substructure content was tested on a curated database of 357 SAD cases with useful anomalous signal. The prior estimates of substructure content are highly correlated to the content determined by phasing calculations, with a correlation coefficient (on a log-log basis) of 0.72.

Advances in long-wavelength native phasing at X-ray free-electron lasers.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

Crystal structure of the periplasmic sensor domain of histidine kinase VbrK suggests indirect sensing of β-lactam antibiotics

Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to β-lactam antibiotics through activating a β-lactamase gene. Its periplasmic sensor domain was previously suggested to detect β-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrKSD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrKSD is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of β-lactam antibiotics to VbrKSD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrKSD did not show any significant binding to β-lactam antibiotics. Therefore, we propose that the detection of β-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.

Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing.

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

Bayesian machine learning improves single-wavelength anomalous diffraction phasing.

Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(-) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.

Development of “Parameter space screening”-based single-wavelength anomalous diffraction phasing and structure determination pipeline

In this paper, we present a highly efficient structure determination pipeline software suite (X2DF) that is based on the “Parameter space screening” method, by combining the popular crystallographic structure determination programs and high-performance parallel computing. The phasing method employed in X2DF is based on the single-wavelength anomalous diffraction (SAD) theory. In the X2DF, the choice of crystallographic software, the input parameters to this software and the results display layout, are all parameters which users can select and screen automatically. Users may submit multiple structure determination jobs each time, and each job uses a slightly different set of input parameters or programs. Upon completion, the results of the calculation performed can be displayed, harvested, and analyzed by using the graphical user interface (GUI) of the system. We have applied the X2DF successfully to many cases including the cases that the structure solutions fail to be yielded by using manual approaches.

Cadmium SAD phasing at CuKα wavelength

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.

Choosing your (Friedel) mates wisely: grouping data sets to improve anomalous signal.

Single-wavelength anomalous diffraction (SAD) phasing from multiple crystals can be especially challenging in samples with weak anomalous signals and/or strong non-isomorphism. Here, advantage is taken of the combinatorial diversity possible in such experiments to study the relationship between merging statistics and downstream metrics of phasing signals. It is furthermore shown that a genetic algorithm (GA) can be used to optimize the grouping of data sets to enhance weak anomalous signals based on these merging statistics.

Cadmium SAD phasing at CuKα wavelength.

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.

Utilizing guanine-coordinated Zn2+ ions to determine DNA crystal structures by single-wavelength anomalous diffraction.

The experimental phase determination of crystal structures of nucleic acids and nucleic acid-ligand complexes would benefit from a facile method. Even for double-stranded DNA, software-generated models are generally insufficiently accurate to serve as molecular replacement search models, necessitating experimental phasing. Here, it is demonstrated that Zn2+ ions coordinated to the N7 atom of guanine bases generate sufficient anomalous signal for single-wavelength anomalous diffraction (SAD) phasing of DNA crystal structures. Using zinc SAD, three crystal structures of double-stranded DNA oligomers, 5'-AGGGATCCCT-3', 5'-GGGATCCC-3' and 5'-GAGGCCTC-3', were determined. By determining the crystal structure of one of these oligomers, GAGGCCTC, in the presence of Mg2+ instead of Zn2+, it was demonstrated that Zn2+ is not structurally perturbing. These structures allowed the analysis of structural changes in the DNA on the binding of analogues of the natural product mithramycin to two of these oligomers, AGGGATCCCT and GAGGCCTC. Zinc SAD may become a routine approach for determining the crystal structures of nucleic acids and their complexes with small molecules.

Fast and accurate data collection for macromolecular crystallography using the JUNGFRAU detector

The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources.

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica

A novel galactose-specific lectin, AJLec (18.5 kDa), was isolated from the sea anemone, Anthopleura japonica. AJLec was characterized using the hemagglutination assay, isothermal titration calorimetry (ITC), and glycoconjugate microarray analysis and we found that AJLec has a specificity for galactose monomers and β-linked terminal galactose residues in complex carbohydrates, but not for N-acetylgalactosamine (GalNAc), which is commonly recognized by galactose-binding lectins. The primary structure of AJLec did not show homology with known lectins, and a crystal structural analysis also revealed a unique homodimeric structure. The crystal structure of AJLec complexed with lactose was solved by measuring the sulfur single-wavelength anomalous diffraction (S-SAD) phasing with an in-house Cu Kα source method. This analysis revealed that the galactose residue in lactose was recognized via its O2, O3, and O4 hydroxyl groups and ring oxygen by calcium coordination and two hydrogen bonds with residues in the carbohydrate-binding site, which demonstrated strict specificity for the β-linked terminal galactose in this lectin.

Maximum-likelihood determination of anomalous substructures.

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.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Å wavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routine de novo structural determination of biomacromolecules.

Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity.

SAD phasing: History, current impact and future opportunities

Single wavelength anomalous diffraction (SAD) can trace its beginnings to the early 1950s. Researchers at the time recognized that SAD offers some unique features that might be advantageous for crystallographic phasing, despite the fact that at that time recording accurate SAD data was problematic. In this review we will follow the trail from those early days, highlighting key advances in the field and interpreting them in terms on how they stimulated continued phasing development that produced the theoretical foundation for the routine macromolecular structure determination by SAD today. The technological advances over the past three decades in both hardware and software, which played a significant role in making SAD phasing a ‘first choice method’, will also be described.