Single-wavelength Anomalous Diffraction Data Research Articles

Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment.

To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F-) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.

Extending the novel |ρ|-based phasing algorithm to the solution of anomalous scattering substructures from SAD data of protein crystals.

Owing to the importance of the single-wavelength anomalous diffraction (SAD) technique, the recently developed |ρ|-based phasing algorithm (SM,|ρ|) incorporating the inner-pixel preservation (ipp) procedure [Rius & Torrelles (2021). Acta Cryst A77, 339-347] has been adapted to the determination of anomalous scattering substructures and its applicability tested on a series of 12 representative experimental data sets, mostly retrieved from the Protein Data Bank. To give an idea of the suitability of the data sets, the main indicators measuring their quality are also given. The dominant anomalous scatterers are either SeMet or S atoms, or metals/clusters incorporated by soaking. The resulting SAD-adapted algorithm solves the substructures of the test protein crystals quite efficiently.

Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing.

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects β-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Å using wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.

Substructure determination using phase-retrieval techniques.

Thus far, the application of phase-retrieval methods in crystallography has mainly been aimed at variants of charge flipping or structure-factor flipping. In this work, the relaxed averaged alternating reflections (RAAR) algorithm is applied to determine anomalously scattering substructures from single-wavelength anomalous diffraction (SAD) data of macromolecules. The algorithm has been implemented in a new program, PRASA, and has been shown to significantly outperform charge flipping in determining anomalously scattering substructures on a test sample of 169 SAD data sets with resolutions up to 3.88 Å.

A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model.

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F1-ATPase data set and a 4.5 Å resolution SecYEG-SecA complex data set. All of the models were automatically built by the method to Rfree values of between 28.9 and 39.9% and were free from the initial model bias.

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.

Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca(2+)-calmodulin.

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca(2+)-CaM, but no assignment of these features has been made. In addition, Ca(2+)-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca(2+)-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca(2+)-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca(2+)-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca(2+)-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca(2+)-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing.

Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.

Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34.

The phage-proximal part of the long tail fibres of bacteriophage T4 consists of a trimer of the 1289 amino-acid gene product 34 (gp34). Different carboxy-terminal parts of gp34 have been produced and crystallized. Crystals of gp34(726-1289) diffracting X-rays to 2.9 Å resolution, crystals of gp34(781-1289) diffracting to 1.9 Å resolution and crystals of gp34(894-1289) diffracting to 3.0 and 2.0 Å resolution and belonging to different crystal forms were obtained. Native data were collected for gp34(726-1289) and gp34(894-1289), while single-wavelength anomalous diffraction data were collected for selenomethionine-containing gp34(781-1289) and gp34(894-1289). For the latter, high-quality anomalous signal was obtained.

Crystallization and preliminary X-ray diffraction studies of D-threo-3-hydroxyaspartate dehydratase isolated from Delftia sp. HT23.

D-threo-3-Hydroxyaspartate dehydratase (D-THA DH) isolated from the soil bacterium Delftia sp. HT23 is a novel enzyme consisting of 380 amino-acid residues which catalyzes the conversion of D-threo-3-hydroxyaspartate to oxaloacetate and ammonia. D-THA DH also catalyzes the dehydration of L-threo-3-hydroxyaspartate, L-erythro-3-hydroxyaspartate and D-serine. The amino-acid sequence of D-THA DH shows significant similarity to that of two eukaryotic D-serine dehydratases derived from Saccharomyces cerevisiae and chicken kidney. D-THA DH is classified into the fold-type III group of pyridoxal enzymes and is the first example of a fold-type III dehydratase derived from a prokaryote. Overexpression of recombinant D-THA DH was carried out using a Rhodococcus erythropolis expression system and the obtained protein was subsequently purified and crystallized. The crystals of D-THA DH belonged to space group I4₁22, with unit-cell parameters a=b=157.3, c=157.9 Å. Single-wavelength anomalous diffraction data were collected to a resolution of 2.0 Å using synchrotron radiation at the wavelength of the Br K absorption edge.

Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa.

The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91-319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR_91-319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-L-methionine-labelled crystals, revealing the space group to be P6(5)22, with unit-cell parameters a = b = 116-120, c = 115-117 Å.

Recent advances in theCRANKsoftware suite for experimental phasing

For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/).

Reduction of density-modification bias by β correction.

Density modification often suffers from an overestimation of phase quality, as seen by escalated figures of merit. A new cross-validation-based method to address this estimation bias by applying a bias-correction parameter 'β' to maximum-likelihood phase-combination functions is proposed. In tests on over 100 single-wavelength anomalous diffraction data sets, the method is shown to produce much more reliable figures of merit and improved electron-density maps. Furthermore, significantly better results are obtained in automated model building iterated with phased refinement using the more accurate phase probability parameters from density modification.

New expression of bimodal phase distributions in direct-method phasing of protein single-wavelength anomalous diffraction data

One of the essential points of the direct-method single-wavelength anomalous diffraction (SAD) phasing for proteins is to express the bimodal SAD phase distribution by the sum of two Gaussian functions peaked respectively at φ″h +|Δφh| and φ″h−|Δφh|. The probability for Δφh being positive (P+) can be derived based on the Cochran distribution in direct methods. Hence the SAD phase ambiguity can be resolved by multiplying the Gaussian function peaked at φ″h+|Δφh| with P+ and multiplying the Gaussian function peaked at φ″h−|Δφh| with P− ( = 1 − P+). The direct-method SAD phasing has been proved powerful in breaking SAD phase ambiguities, in particular when anomalous-scattering signals are weak. However, the approximation of bimodal phase distributions by the sum of two Gaussian functions introduces considerable errors. In this paper we show that a much better approximation can be achieved by replacing the two Gaussian functions with two von Mises distributions. Test results showed that this leads to significant improvement on the efficiency of direct-method SAD-phasing.

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

Crystal Structure of SmcR, a Quorum-sensing Master Regulator of Vibrio vulnificus, Provides Insight into Its Regulation of Transcription

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 Å resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr805, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.

Use of iterative OASIS with low resolution SAD laboratory X-ray source (Cr Kα) data sets

Anomalous scattering methods are widely used for phasing macromolecular structures. OASIS program works on a direct methods procedure and is used to break the phase ambiguity intrinsic in a single wavelength anomalous diffraction (SAD) experiment. An iterative phasing/model-building procedure for processing SAD data includes the initial direct method phasing of SAD data, density modification, automatic model building and direct methods phasing of SAD data with feed back from partial structure information. This dual-space procedure has been tested on two experimental Cr Kα SAD data sets, 2.3 A for S-SAD and 2.4 A for I-SAD, of an enzyme glucose isomerase with an approximate molecular weight of 44 kDa (388 residues). HYSS in PHENIX was used to locate the anomalous scatterers for both the data sets (11 sulfurs for 2.3 A data and 9 iodines for 2.4 A data). Phasing and density modification were carried out using OASIS, DM and model building was then carried out using ARP/wARP. Two iterative cycles were necessary for the successful structure solution and refinement of the 2.3 A data set which built 384 residues out of 388. Regular ARP/wARP failed for the 2.4 A resolution data and hence the modified version of REFMAC5 was used in ARP/wARP. A successful model could be built after three iterative cycles of OASIS using this modified version which built 382 residues out of 388 residues. Minimal manual model building was required at this stage and the structure determination was completed using regular REFMAC5. All the computations mentioned here were carried out using Pentium IV PC.

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl(2) (2.1 A; R = 18.2%, R(free) = 21.9%), and in complex with glutamate, MgCl(2), and ADP (2.7 A; R = 19.0%, R(free) = 24.2%). Inspection of these structures reveals an unusual binding pocket for the alpha-carboxylate of the glutamate substrate and an ATP-independent Mg(2+) coordination site, clarifying the Mg(2+) dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

Direct-method SAD phasing of proteins enhanced by the use of intrinsic bimodal phase distributions in the subsequent phase-improvement process

A modified SAD (single-wavelength anomalous diffraction) phasing algorithm has been introduced in the latest version of the program OASIS. In addition to direct-method phases and figures of merit, Hendrickson-Lattman coefficients that correspond to the original unresolved bimodal phase distributions are also output and used in subsequent phase-improvement procedures in combination with the improved phases. This provides the possibility of rebreaking the SAD phase ambiguity using the ever-improving phases resulting from the phase-improvement process. Tests using experimental SAD data from six known proteins showed that in all cases the new treatment produced significant improved results.