Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

The galactoside 2-α-L-fucosyltransferase FUT1 fromArabidopsis thaliana: crystallization and experimental MAD phasing

The plant cell wall is a complex network of polysaccharides made up of cellulose, hemicelluloses and pectins. Xyloglucan (XyG), which is the main hemicellulosic component of dicotyledonous plants, has attracted much attention for its role in plant development and for its many industrial applications. The XyG-specific fucosyltransferase (FUT1) adds a fucose residue from GDP-fucose to the 2-O position of the terminal galactosyl residues on XyG side chains. Recombinant FUT1 fromArabidopsis thalianawas crystallized in two different crystal forms, with the best diffracting crystals (up to 1.95 Å resolution) belonging to the monoclinic space groupP21, with unit-cell parametersa= 87.6,b= 84.5,c = 150.3 Å, β = 96.3°.Ab initiophases were determined using a two-wavelength anomalous dispersion experiment on a tantalum bromide-derivatized crystal with data collected at the rising and descending inflection points of the Ta white line. An interpretable electron-density map was obtained after elaborate density modification. Model completion and structural analysis are currently under way.

Crystallization and preliminary X-ray analysis of the C-terminal fragment of Ski7 fromSaccharomyces cerevisiae

Ski7 (superkiller protein 7) plays a critical role in the mRNA surveillance pathway. The C-terminal fragment of Ski7 (residues 520–747) fromSaccharomyces cerevisiaewas heterologously expressed inEscherichia coliand purified to homogeneity. It was successfully crystallized and preliminary X-ray data were collected to 2.0 Å resolution using synchrotron radiation. The crystal belonged to a trigonal space group, eitherP3121 orP3221, with unit-cell parametersa=b= 73.5,c= 83.6 Å. The asymmetric unit contains one molecule of the C-terminal fragment of Ski7 with a corresponding crystal volume per protein mass (VM) of 2.61 Å3 Da−1and a solvent content of 52.8% by volume. The mergingRfactor is 6.6%. Structure determination by MAD phasing is under way.

Big Methods for Small Molecules

Since the early 70's, multiple wavelength experiments have been used to determine phases of proteins containing anomalous scatterers. The small molecule single crystal beamline, I19,[1] at Diamond Light Source, is designed to carry out single crystal anomalous dispersion studies using tunable wavelength. These experiments can differentiate between oxidation states; discriminate between atoms with near-identical X-ray scattering factors; and solve the phase problem for very low resolution X-ray data. We describe the application of MAD phasing (Karle and Hendrickson [2]) to determine the structure of large `small molecules' where only low-resolution data is available. Initial studies were carried out on a known, (well diffracting) centrosymmetric bromide containing compound. The wavelength dependence of the anomalous signal from the bromide was calculated from fluorescence absorption data in DetOx.[3] Datasets were then collected at 4 wavelengths chosen to maximize differences in the anomalous signal. Using the MAD phasing equations we obtained estimates for the anomalous scattering contribution from all atoms in the structure and a phase difference between that and the normal scattering component. This allowed us to reduce noise in the Patterson map and locate only the heavy atom scatterers. We then use phase estimates from the heavy atom substructure to locate the rest of the atoms. Initial proof of concept experiments will now be extended to larger structures where data is not of sufficient resolution to be solved by direct methods alone.

Structure and possible mechanism of the CcbJ methyltransferase fromStreptomyces caelestis

TheS-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ fromStreptomyces caelestiscatalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex withS-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ–SAM–substrate and CcbJ–SAH–product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H+ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.

Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteinsviaMAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage.