Single Isomorphous Replacement With Anomalous Scattering Research Articles

De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography.

During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection.

A case study on the treatment of protein SIRAS data.

A case study has been made on the treatment of the SIRAS (single isomorphous replacement with anomalous scattering) data of the originally unknown protein LegC3N. An alternative treatment has been proposed which led to improved results in this particular test case. The treatment involves iterative direct-method SAD (single-wavelength anomalous diffraction) phasing and direct-method-aided model completion, both of which are implanted in the IPCAS (Iterative Protein Crystal-structure Automatic Solution) pipeline. Apart from the experimental data, a simulated SIRAS data set for LegC3N with the derivative data truncated to 5.0 Å resolution has also been tested. SAD phasing and phase/model extension in PHENIX without direct methods failed to solve the structure using these simulated SIRAS data. However, the procedure proposed here involving direct methods in both SAD phasing and phase/model extension led to a nearly complete structure model. This shows the potential ability of treating SIRAS data with a derivative diffracting to lower resolution.

Alternative phasing method in macromolecular crystallography

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].

Crystallization, X-ray diffraction analysis and SIRAS phasing of human α-L-iduronidase

Human lysosomal α-L-iduronidase, whose deficiency causes mucopolysaccharidosis type I, was crystallized using sodium/potassium tartrate and polyethylene glycol 3350 as a precipitant. Using synchrotron radiation, a native data set was collected from a single crystal at 100 K to 2.3 Å resolution. The crystal belonged to space group R3 with unit-cell dimensions of a=b=259.22, c=71.83 Å. To obtain the phase information, mercury-derivative crystals were prepared and a single-wavelength anomalous dispersion (SAD) data set was collected at the Hg peak wavelength. Phase calculation with the single isomorphous replacement with anomalous scattering (SIRAS) method successfully yielded an interpretable electron-density map.

Homophilic Adhesion Mechanism of Neurofascin, a Member of the L1 Family of Neural Cell Adhesion Molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

Structure of the C-terminal domain of nsp4 from feline coronavirus

Coronaviruses are a family of positive‐stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C‐terminal domain (∼100 amino‐acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library‐based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet‐substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS‐derived composite model. The structure consists of a single domain with a predominantly α‐helical content displaying a unique fold that could be engaged in protein–protein interactions.

Crystal structure of a fungal protease inhibitor from Antheraea mylitta

Indian tasar silk is produced by a wild insect called Antheraea mylitta. Insects do not have any antigen-antibody mediated immune system like vertebrates but they produce a wide variety of effector proteins and peptides possessing potent antifungal and antibacterial activity to combat microbial attack. Antheraea mylitta expresses a fungal protease inhibitor AmFPI-1, in the hemolymph that inhibits alkaline protease of Aspergillus oryzae for protection against fungal infection. AmFPI-1 is purified from the hemolymph, crystallized and the structure is solved using the single isomorphous replacement with anomalous scattering (SIRAS) method to a resolution of 2.1 A. AmFPI-1 is a single domain protein possessing a unique fold that consists of three helices and five beta strands stabilized by a network of six disulfide bonds. The reactive site of AmFPI-1 is located in the loop formed by residues 46-66, wherein Lys54 is the P(1) residue. Superimposition of the loop with reactive sites of other canonical protease inhibitors shows that reactive site conformation of AmFPI-1 is similar to them. The structure of AmFPI-1 provides a framework for the docking of a 1:1 complex between AmFPI-1 and alkaline protease. This study addresses the structural basis of AmFPI-1's specificity towards a fungal serine protease but not to mammalian trypsin and may help in designing specific inhibitors against fungal proteases.

Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli.

The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222(1); the Ta6Br12(2+) cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta6Br12(2+)-derivatized structure to 5 A resolution. Many of the Ta6Br12(2+)-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006), J. Biol. Chem. 281, 1532-1546].

Structural and transcriptional analyses of a purine nucleotide-binding protein from Pyrococcus furiosus: a component of a novel, membrane-bound multiprotein complex unique to this hyperthermophilic archaeon

The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.

The molecular structure of Rv1873, a conserved hypothetical protein from Mycobacterium tuberculosis, at 1.38 A resolution.

The X-ray crystal structure of the gene product encoded by open reading frame Rv1873 of Mycobacterium tuberculosis has been determined by single isomorphous replacement with anomalous scattering (SIRAS) phasing techniques at 1.38 A resolution from monoclinic crystals with unit-cell parameters a = 33.44, b = 31.63, c = 53.19 A, beta = 90.8 degrees. The 16.2 kDa Rv1873 is a monomer that adopts a primarily alpha-helical fold with limited structural similarity to previously determined tertiary structures. It has been annotated as a conserved hypothetical protein of unknown function and is classified by the Clusters of Orthologous Groups (COG) database as belonging to COG5579. The three-dimensional structure of the Rv1873 gene product reveals limited similarity to a repeated motif that is found in a variety of other proteins. While not a novel fold, it serves as a model for orthologues predicted to be related by sequence and it is hoped that knowledge of the structure of Rv1873 will aid in determining a possible function for this protein.

Anomalous diffraction at ultra-high energy for protein crystallography

Single-wavelength anomalous diffraction (SAD), multiwavelength anomalous diffraction (MAD) and single isomorphous replacement with anomalous scattering (SIRAS) phasing at ultra-high X-ray energy, 55 keV, are used successfully to determine a high-quality and high-resolution experimental electronic density map of hen egg-white lysozyme, a model protein. Several combinations, between single- and three-wavelength, with native data were exploited to demonstrate that standard phasing procedures with standard equipment and software can successfully be applied to three-dimensional crystal structure determination of a macromolecule, even at these very short wavelengths. For the first time, a high-quality three-dimensional molecular structure is reported from SAD phasing with ultra-high-energy X-rays. The quality of the crystallographic data and the experimental electron density maps meet current standards. The 2.7% anomalous signal from three Ho atoms, at the HoKedge, was sufficient to obtain a remarkable electron density and build the first lanthanide structure for HEWL in its entirety.

Structural Analysis of N-acetylglucosamine-6-phosphate Deacetylase Apoenzyme from Escherichia coli

We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.

Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides.

A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 A resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 A. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5 A3 Da(-1)). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 A resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.

Structure of the N-Terminal Domain of the Adenylyl Cyclase-Associated Protein (CAP) from Dictyostelium discoideum

Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51–226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an α helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1–226), CAP(255–464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

Crystal Structure of a Transcarbamylase-like Protein from the Anaerobic Bacterium Bacteroides fragilis at 2.0 Å Resolution

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4 32 12 ( a= b=153.4 Å, c=94.8 Å). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 Å resolution to an R-factor of 20.6% ( R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel β-sheet surrounded by α-helices and loops with α/β topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif.

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 Å resolution

Background: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. Results: The crystal structure of Omp32 was determined to a resolution of 2.1 Å using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded β barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within β-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with β-strand 1 of the porin. Conclusions: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.

Novel approach to phasing proteins: derivatization by short cryo-soaking with halides.

A quick (less than 1 min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30 kDa.

Freeze-Trapping Isomorphous Xenon Derivatives of Protein Crystals

A simple and efficient method to prepare isomorphous derivatives of protein crystals with xenon as a heavy atom is described. The method consists of exposing a crystal to xenon gas of pressures above 5 atm (~ 0.5 MPa) for several minutes and subsequently shock-freezing the crystal to immobilize the xenon dissolved in the mother liquor and bound to the protein. Diffraction data can the be collected with the established techniques of protein cryocrystallography. Two types of high-pressure device are described to expose a protein crystal to the required xenon pressure, permitting rapid freeze-quenching after xenon exposure. One of these devices can be used for gas pressures up to and exceeding 5.0 MPa, with a gas consumption of a few millilitres of uncompressed gas. The technique has been tested with monoclinic crystals of sperm-whale metmyoglobin, which has four xenon binding sites. The results of these experiments are described and discussed. Potential applications of this technique include-besides the classical multiwavelength anomalous diffraction (MAD) or single isomorphous replacement with anomalous scattering (SIRAS) experiments-the derivation of low-angle phase information by modifying the electron density of the solvent regions within the crystal.