Single-wavelength Anomalous Dispersion Phasing Research Articles

Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase

Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP.

Radiation damage to biological samples: still a pertinent issue.

An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques. For X-ray diffraction, an in-depth study of multi-crystal small-wedge data collection single-wavelength anomalous dispersion phasing protocols is presented, concluding that an absorbed dose of 5 MGy per crystal was optimal to allow reliable phasing. For small-angle X-ray scattering, experiments are reported that evaluate the efficacy of three radical scavengers using a protein designed to give a clear signature of damage in the form of a large conformational change upon the breakage of a disulfide bond. The use of X-rays to induce OH radicals from the radiolysis of water for X-ray footprinting are covered in two papers. In the first, new developments and the data collection pipeline at the NSLS-II high-throughput dedicated synchrotron beamline are described, and, in the second, the X-ray induced changes in three different proteins under aerobic and low-oxygen conditions are investigated and correlated with the absorbed dose. Studies in XFEL science are represented by a report on simulations of ultrafast dynamics in protic ionic liquids, and, lastly, a broad coverage of possible methods for dose efficiency improvement in modalities using electrons is presented. These papers, as well as a brief synopsis of some other relevant literature published since the last Journal of Synchrotron Radiation Special Issue on Radiation Damage in 2019, are summarized below.

De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing.

Single-wavelength anomalous dispersion (SAD)-phasing using sulfur as the unique anomalous scatterer is a powerful method to solve the phase problem in protein crystallography. However, it is not yet widely used by non-expert crystallographers. We report here the structure determination of the double stranded RNA binding domain of human dihydrouridine synthase using the sulfur-SAD method and highly redundant data collected at 1.8 ​Å (“off-edge”), at which the estimated overall anomalous signal was 1.08%. High multiplicity data were collected on a single crystal rotated along the ϕ or ω axis at different κ angles, with the primary beam intensity being attenuated from 50% to 95%, compared to data collection at 0.98 ​Å, to reduce radiation damage. SHELXD succeeded to locate 14 out 15 sulfur sites only using the data sets recorded with highest beam attenuation, which provided phases sufficient for structure solving. In an attempt to stimulate the use of sulfur-SAD phasing by a broader community of crystallographers, we describe our experimental strategy together with a compilation of previous successful cases, suggesting that sulfur-SAD phasing should be attempted for determining the de novo structure of any protein with average sulfur content diffracting better than 3 ​Å resolution.

The predictive power of data-processing statistics.

This study describes a method to estimate the likelihood of success in determining a macromolecular structure by X-ray crystallography and experimental single-wavelength anomalous dispersion (SAD) or multiple-wavelength anomalous dispersion (MAD) phasing based on initial data-processing statistics and sample crystal properties. Such a predictive tool can rapidly assess the usefulness of data and guide the collection of an optimal data set. The increase in data rates from modern macromolecular crystallography beamlines, together with a demand from users for real-time feedback, has led to pressure on computational resources and a need for smarter data handling. Statistical and machine-learning methods have been applied to construct a classifier that displays 95% accuracy for training and testing data sets compiled from 440 solved structures. Applying this classifier to new data achieved 79% accuracy. These scores already provide clear guidance as to the effective use of computing resources and offer a starting point for a personalized data-collection assistant.

A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion

Escherichia coli (E. coli), one of the most frequently used host for the expression of recombinant proteins, is often affected by the toxic effect of the exogenous proteins that is required to express. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multi-functional protein that has been used as a control marker for basal function and it is known to undergo cysteine oxidation under different types of cellular stress. Here, we report the 3D structure of the endogenous GAPDH purified from stressed E. coli cells expressing a eukaryotic protein. The structure was solved at 1.64 Å using single-wavelength anomalous dispersion (SAD) phasing with a selenium-modified enzyme. Interestingly, each GAPDH monomer contains a molecule of glyceraldehyde-3 phosphate in a non-previously identified site. Furthermore, the catalytic Cys149 is covalently attached to a ~300 Da molecule, possibly glutathione. This modification alters the conformation of an adjacent alpha helix in the catalytic domain, right opposite to the NAD+ binding site. The conformation of the alpha helix is stabilized after soaking the crystals with NAD+. These results exemplify the effects that the overexpression of an exogenous protein has over the host proteins and sheds light on the structural changes that large oxidant molecules on the catalytic cysteine produce for the GAPDH enzyme.

Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99-107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels-Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels-Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399.

Phasing with calcium at home.

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.

Making routine native SAD a reality: lessons from beamline X06DA at the Swiss Light Source.

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.

In-house high-energy-remote SAD phasing using the magic triangle: how to tackle the P1 low symmetry using multiple orientations of the same crystal of human IBA57 to increase the multiplicity.

This article describes the approach used to solve the structure of human IBA57 in-house by 5-amino-2,4,6-triiodoisophthalic acid (I3C) high-energy-remote single-wavelength anomalous dispersion (SAD) phasing. Multiple orientations of the same triclinic crystal were exploited to acquire sufficient real data multiplicity for phasing. How the collection of an in-house native data set and its joint use with the I3C derivative through a SIRAS approach decreases the data multiplicity needed by almost 50% is described. Furthermore, it is illustrated that there is a clear data-multiplicity threshold value for success and failure in phasing, and how adding further data does not significantly affect substructure solution and model building. To our knowledge, this is the only structure present in the PDB that has been solved in-house by remote SAD phasing in space group P1 using only one crystal. All of the raw data used, derived from the different orientations, have been uploaded to Zenodo in order to enable software developers to improve methods for data processing and structure solution, and for educational purposes.

Crystal structure of native α-L-rhamnosidase from Aspergillus terreus.

α-L-Rhamnosidases cleave terminal nonreducing α-L-rhamnosyl residues from many natural rhamnoglycosides. This makes them catalysts of interest for various biotechnological applications. The X-ray structure of the GH78 family α-L-rhamnosidase from Aspergillus terreus has been determined at 1.38 Å resolution using the sulfur single-wavelength anomalous dispersion phasing method. The protein was isolated from its natural source in the native glycosylated form, and the active site contained a glucose molecule, probably from the growth medium. In addition to its catalytic domain, the α-L-rhamnosidase from A.terreus contains four accessory domains of unknown function. The structural data suggest that two of these accessory domains, E and F, might play a role in stabilizing the aglycon portion of the bound substrate.

The signal-to-noise ratio in SAD experiments

ABSTRACTDe novo single-wavelength anomalous dispersion (SAD) determination of macromolecular structures requires accurate measurement of anomalous signal from naturally occurring (S, P, Ca, etc.) or incorporated (Se, Hg, etc.) anomalous scatterers. The quality and the level of the anomalous signal in SAD datasets can be assessed using, as an indicator, the average anomalous signal-to-noise level ⟨|ΔIanom|/σ(ΔI)⟩ based on intensities or ⟨|ΔFanom|/σ(ΔF)⟩ based on amplitudes. The value for the average anomalous signal-to-noise for a successful SAD experiment is a matter of debate given its high variability from one SAD experiment to another. We present a mathematical model relating the ⟨|ΔIanom|/σ(ΔI)⟩ to the overall final ⟨I/σ(I)⟩, necessary for successful SAD phasing. By statistical analysis of the data from 115 successful native SAD experiments, as available in the PDB, we show that the experimental values of ⟨|ΔIanom|/σ(ΔI)⟩ follow a Gamma distribution with an average value of 1.2 ± 0.3. This mathematical model allows a-priori prediction of the overall ⟨I/σ(I)⟩, necessary for successful SAD phasing given the description of the sample expressed. The results reported here are of general applicability to any phasing experiment involving the measurement of anomalous signal from any anomalous scatterer or X-ray source, including X-FEL data.

Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches\u2019 Broom Disease of Cacao

The pathogenic fungi Moniliophthora perniciosa causes Witches’ Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.

Rapid cadmium SAD phasing at the standard wavelength (1 Å).

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Å is demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP-divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths (i.e. no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing.

Rapid experimental SAD phasing and hot-spot identification with halogenated fragments.

Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a 'magic bullet' that is capable of binding at many of the ligand 'hot spots' found in HIV-1 reverse transcriptase (RT). The binding locations can be in pockets that are 'hidden' in the unliganded crystal form, allowing rapid identification of these sites for in silico screening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K) by single-wavelength anomalous dispersion (SAD) from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals.

A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys.).

The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu (“EF-Pyl”). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg2+, MM1309·GDP·Mg2+, and MM1309·GMPPNP·Mg2+ provided dissociation constants of 0.43, 26.2, and 222.2 μM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain–domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNACys and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNACys.

Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

Refined structures of mouse P‐glycoprotein

The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Å resolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Å resolution with a 9.4 and 8.1% decrease in Rwork and Rfree, respectively, (Rwork = 21.2%, Rfree = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Å from their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.

The Crystal Structure of the Core Domain of a Cellulose Induced Protein (Cip1) from Hypocrea jecorina, at 1.5 Å Resolution

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.

ChipX: A Novel Microfluidic Chip for Counter-Diffusion Crystallization of Biomolecules and in Situ Crystal Analysis at Room Temperature

Microfluidic technology has opened new possibilities for the crystallization of biological macromolecules during the past decade. Microfluidic systems offer numerous advantages over conventional crystal growth methods. They enable easy handling of nanovolumes of solutions, extreme miniaturization, and parallelization of crystallization assays, especially for high-throughput screening applications. Our goal was to design a versatile, low cost, and easy-to-use crystallization chip based on counter-diffusion that is compatible with on-chip crystallographic characterization. The ChipX is a microfluidic chip made of cyclic olefin copolymer. It was used to grow crystals of biomolecules and perform complete X-ray diffraction analyses on synchrotron sources. Our results demonstrate that accurate crystallographic data can be collected at room temperature directly from ChipX microfluidic devices for both experimental single-wavelength anomalous dispersion phasing and structure refinement.

In-house zinc SAD phasing at Cu Kα edge.

De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that inhouse Zn-SAD phasing can be widely applicable to structure determination.