Multiple-wavelength Anomalous Dispersion Research Articles

The extracellular region of bovine milk butyrophilin exhibits closer structural similarity to human myelin oligodendrocyte glycoprotein than to immunological BTN family receptors.

Bovine butyrophilin (BTN1A1) is an abundant type I transmembrane glycoprotein exposed on the surface of milk fat globules. We have solved the crystal structure of its extracellular region via multiple wavelength anomalous dispersion after incorporation of selenomethionine into the bacterially produced protein. The butyrophilin ectodomain exhibits two subdomains with immunoglobulin fold, each comprising a β-sandwich with a central disulfide bridge as well as one N-linked glycosylation. The fifth Cys residue at position 193 is unpaired and prone to forming disulfide crosslinks. The apparent lack of a ligand-binding site or receptor activity suggests a function predominantly as hydrophilic coat protein to prevent coagulation of the milk fat droplets. While there is less structural resemblance to members of the human butyrophilin family such as BTN3A, which play a role as immune receptors, the N-terminal bovine butyrophilin subdomain shows surprising similarity to the human myelin oligodendrocyte glycoprotein, a protein exposed on the surface of myelin sheaths. Thus, our study lends structural support to earlier hypotheses of a correlation between the consumption of cow milk and prevalence of neurological autoimmune diseases and may offer guidance for the breeding of cattle strains that express modified butyrophilin showing less immunological cross-reactivity.

SPIND-TC: an indexing method for two-color X-ray diffraction data.

Recent developments of two-color operation modes at X-ray free-electron laser facilities provide new research opportunities, such as X-ray pump/X-ray probe experiments and multiple-wavelength anomalous dispersion phasing methods. However, most existing indexing methods were developed for indexing diffraction data from monochromatic X-ray beams. Here, a new algorithm is presented for indexing two-color diffraction data, as an extension of the sparse-pattern indexing algorithm SPIND, which has been demonstrated to be capable of indexing diffraction patterns with as few as five peaks. The principle and implementation of the two-color indexing method, SPIND-TC, are reported in this paper. The algorithm was tested on both simulated and experimental data of protein crystals. The results show that the diffraction data can be accurately indexed in both cases. Source codes are publicly available at https://github.com/lixx11/SPIND-TC.

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.

Two-colour serial femtosecond crystallography dataset from gadoteridol-derivatized lysozyme for MAD phasing

We provide a detailed description of a gadoteridol-derivatized lysozyme (gadolinium lysozyme) two-colour serial femtosecond crystallography (SFX) dataset for multiple wavelength anomalous dispersion (MAD) structure determination. The data was collected at the Spring-8 Angstrom Compact free-electron LAser (SACLA) facility using a two-colour double-pulse beam to record two diffraction patterns simultaneously in one diffraction image. Gadolinium lysozyme was chosen as a well-established model system that has a very strong anomalous signal. Diffraction patterns from gadolinium lysozyme microcrystals were recorded to a resolution of 1.9 Å in both colours. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development.

Multi-wavelength anomalous diffraction de novo phasing using a two-colour X-ray free-electron laser with wide tunability

Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage. However, de novo structure determination, i.e., without prior structural knowledge, is complicated by the inherent inaccuracy of serial femtosecond crystallography data. By its very nature, serial femtosecond crystallography data collection entails shot-to-shot fluctuations in X-ray wavelength and intensity as well as variations in crystal size and quality that must be averaged out. Hence, to obtain accurate diffraction intensities for de novo phasing, large numbers of diffraction patterns are required, and, concomitantly large volumes of sample and long X-ray free-electron laser beamtimes. Here we show that serial femtosecond crystallography data collected using simultaneous two-colour X-ray free-electron laser pulses can be used for multiple wavelength anomalous dispersion phasing. The phase angle determination is significantly more accurate than for single-colour phasing. We anticipate that two-colour multiple wavelength anomalous dispersion phasing will enhance structure determination of difficult-to-phase proteins at X-ray free-electron lasers.

Crystal structure of Aquifex aeolicus gene product Aq1627: a putative phosphoglucosamine mutase reveals a unique C-terminal end-to-end disulfide linkage

The Aq1627 gene from Aquifex aeolicus, a hyperthermophilic bacterium has been cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity and its X-ray crystal structure was determined to 1.3 Å resolution using multiple wavelength anomalous dispersion phasing. The structural and sequence analysis of Aq1627 is suggestive of a putative phosphoglucosamine mutase. The structural features of Aq1627 further indicate that it could belong to a new subclass of the phosphoglucosamine mutase family. Aq1627 structure contains a unique C-terminal end-to-end disulfide bond, which links two monomers and this structural information can be used in protein engineering to make proteins more stable in different applications.

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader.

The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

High-intensity double-pulse X-ray free-electron laser.

The X-ray free-electron laser has opened a new era for photon science, improving the X-ray brightness by ten orders of magnitude over previously available sources. Similar to an optical laser, the spectral and temporal structure of the radiation pulses can be tailored to the specific needs of many experiments by accurately manipulating the lasing medium, that is, the electron beam. Here we report the generation of mJ-level two-colour hard X-ray pulses of few femtoseconds duration with an XFEL driven by twin electron bunches at the Linac Coherent Light Source. This performance represents an improvement of over an order of magnitude in peak power over state-of-the-art two-colour XFELs. The unprecedented intensity and temporal coherence of this new two-colour X-ray free-electron laser enable an entirely new set of scientific applications, ranging from X-ray pump/X-ray probe experiments to the imaging of complex biological samples with multiple wavelength anomalous dispersion.

Expression, purification and crystallization of a novel carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin. The novel CBM-Rf1 has been purified and crystallized. The crystals belonged to the trigonal space group R32:H. The CBM-Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively.

Purification, crystallization and preliminary X-ray crystallographic analysis of the flagellar accessory protein FlaH from the methanogenic archaeon Methanocaldococcus jannaschii.

The flagellar accessory protein FlaH is thought to be one of the essential components of an archaeal motility system. However, to date biochemical and structural information about this protein has been limited. Here, the crystallization of FlaH from the hyperthermophilic archaeon Methanocaldococcus jannaschii is reported. Protein crystals were obtained by the vapour-diffusion method. These crystals belonged to space group P3₁21, with unit-cell parameters a=b=131.42, c=89.35 Å. The initial solution of the FlaH structure has been determined by multiple-wavelength anomalous dispersion phasing using a selenomethionine-derivatized crystal.

MxDC & MxLIVE:Software for Macromolecular Crystallography Experiments at the CLS

The Canadian Macromolecular Crystallography Facility (CMCF) is a suite of two beamlines 08ID-1 and 08B1-1. Beamline 08B1-1 is a bending-magnet beamline for high-throughput macromolecular crystallography enabling Multiple-Wavelength Anomalous Dispersion (MAD) and Single-Wavelength Anomalous Dispersion (SAD) experiments with a high level of automation. We have developed an integrated software system with modules for beamline control, experiment management, and automated data processing for both on-side and remote users. The experiment management module, also known as MxLIVE (Macromolecular Crystallography Laboratory Information Virtual Environment) is responsible for managing the storage of information about samples, sample shipments, experiment requests, experiment results and data sets. It provides a web-based interface for users to submit sample information and experiment requests, track shipments en route to the CLS and review experiment results and data sets as they are completed on site, and for beamline staff to manage Mail-In data acquisition sessions, reducing the need for user travel to the synchrotron. The beamline control module includes a user-friendly interface for data collection, MxDC (Macromolecular Crystallography Data Collector). MxDC is fully integrated with beamline hardware as well as software applications such as MxLIVE and AutoProcess, an innovative data processing pipeline. This makes MxDC a hub for all experiment-focused activities at CMCF beamlines, including sample auto-mounting, centering and screening crystals, diffraction experiments, and automated data reduction.

Structure of the NheA Component of the Nhe Toxin from Bacillus cereus: Implications for Function

The structure of NheA, a component of the Bacillus cereus Nhe tripartite toxin, has been solved at 2.05 Å resolution using selenomethionine multiple-wavelength anomalous dispersion (MAD). The structure shows it to have a fold that is similar to the Bacillus cereus Hbl-B and E. coli ClyA toxins, and it is therefore a member of the ClyA superfamily of α-helical pore forming toxins (α-PFTs), although its head domain is significantly enlarged compared with those of ClyA or Hbl-B. The hydrophobic β-hairpin structure that is a characteristic of these toxins is replaced by an amphipathic β-hairpin connected to the main structure via a β-latch that is reminiscent of a similar structure in the β-PFT Staphylococcus aureus α-hemolysin. Taken together these results suggest that, although it is a member of an archetypal α-PFT family of toxins, NheA may be capable of forming a β rather than an α pore.

Crystallization and preliminary X-ray crystallographic analysis of the amylomaltase from Corynebacterium glutamicum.

Amylomaltase (AM; EC 2.4.1.25) belongs to the 4-α-glucanotransferase group of the α-amylase family. The enzyme can produce cycloamylose or large-ring cyclodextrin through intramolecular transglycosylation or cyclization reactions of α-1,4-glucan. Amylomaltase from the mesophilic bacterium Corynebacterium glutamicum (CgAM) contains extra residues at the N-terminus for which the three-dimensional structure is not yet known. In this study, CgAM was overexpressed and purified to homogeneity using DEAE FF and Phenyl FF columns. The purified CgAM was crystallized by the vapour-diffusion method. Preliminary X-ray data showed that the CgAM crystal diffracted to 1.7 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 73.28, b = 82.61, c = 118.64 Å. To obtain the initial phases, crystals of selenomethionyl-substituted amylomaltase were produced, and multiple-wavelength anomalous dispersion phasing and structure refinement are now in progress.

Structural Characterization of the RLCK Family Member BSK8: A Pseudokinase with an Unprecedented Architecture

Brassinosteroid signaling kinases (BSKs) are plant-specific receptor-like cytoplasmic protein kinases involved in the brassinosteroid signaling pathway. Unlike common protein kinases, they possess a naturally occurring alanine residue at the “gatekeeper” position, as well as other sequence variations. How BSKs activate downstream proteins such as BSU1, as well as the structural consequences of their unusual sequential features, was unclear. We crystallized the catalytic domain of BSK8 and solved its structure by multiple-wavelength anomalous dispersion phasing methods to a resolution of 1.5Å. In addition, a co-crystal structure of BSK8 with 5-adenylyl imidodiphosphate (AMP-PNP) revealed unusual conformational arrangements of the nucleotide phosphate groups and catalytic key motifs, typically not observed for active protein kinases. Sequential analysis and comparisons with known pseudokinase structures suggest that BSKs represent constitutively inactive protein kinases that regulate brassinosteroid signal transfer through an allosteric mechanism.

Crystal Structure of Arabidopsis thaliana Dawdle Forkhead-Associated Domain Reveals a Conserved Phospho-Threonine Recognition Cleft for Dicer-Like 1 Binding

Dawdle (DDL) is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated (FHA) domain at the carboxyl-terminus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7-Å resolution. DDL FHA structure displays a seven-stranded β-sandwich architecture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthamiana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr+3(Ile/Val/Leu/Asp) motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated protein-protein interaction in plant miRNA processing.

Cloning, expression, purification and preliminary X-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages.

Mycobacterium species have developed numerous strategies to avoid the antimycobacterial actions of macrophages, enabling them to survive within the generally inhospitable environment of the cell. The recently identified MSMEG_5817 protein from M. smegmatis is highly conserved in Mycobacterium spp. and is required for bacterial survival in macrophages. Here, the cloning, expression, purification and crystallization of MSMEG_5817 is reported. Crystals of MSMEG_5817 were grown in 1.42 M Li2SO4, 0.1 M Tris-HCl pH 7.7, 0.1 M sodium citrate tribasic dihydrate. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 fromBovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Functional and Structural Characterization of PaeM, a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Crystallization and preliminary X-ray data analysis of a DJ-1 homologue from Arabidopsis thaliana (AtDJ-1D).

A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, β = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.

Crystallization and preliminary X-ray diffraction of the first periplasmic domain of SecDF, a translocon-associated membrane protein, fromThermus thermophilus

A membrane-integrated Sec component, SecDF, associates with the SecYEG protein-conducting channel and facilitates protein secretion and membrane-protein integration. SecDF contains 12 transmembrane helices and two periplasmic domains. The first periplasmic domain (P1) plays an important role in protein translocation. Here, the overexpression, purification and crystallization of the P1 domain of Thermus thermophilus SecDF are reported. The crystals diffracted X-rays to 2.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 161.1, b = 35.8, c = 181.6 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by the multiple-wavelength anomalous dispersion method using selenomethionine-labelled crystals.