Selenomethionine

The overall scoring procedure is in three steps. A starting set of 10–50 trial heavy-atom partial structures are each given raw scores based on each of the four criteria described above and shown in Table 1 ▶. The mean and standard deviation of the raw scores for each criterion are calculated and are then used as a basis for normalizing all these and later raw scores to yield Z scores for each criteria, where the Z score, based on a raw score of A and a mean and standard deviation for the starting set of and , is given by The final score for a heavy-atom solution is the sum of the Z scores for each of the four criteria. To reduce the likelihood of obtaining a high-scoring solution based on just the Patterson, figure of merit or cross-validation difference Fourier Z scores, the final score is adjusted by subtraction of half the differences between each of these and lowest Z score among them.
When the native Fourier is of low quality, the corresponding score is not of significant utility. To reduce the contribution of the scoring from the native Fourier in cases where it is not expected to be of value, we limit the Z score for the native Fourier to a maximum value depending on the figure of merit of the map. The maximum value is set at the value obtained for cases with the corresponding figure of merit in a series of model calculations we carried out using selenomethionine MAD data and the gene 5 protein atomic model (Terwilliger & Berendzen, 1999 ▶ ; Skinner et al., 1994 ▶ ). These model cases resulted in the approximate relation where m is the average figure of merit of the phase calculation. That is, for a map with a figure of merit of 0.4, the maximum Z score allowed for this criteria would be just 0.6, while for a map with a figure of merit of 0.6 it could be as high as 2.7.

The American Type Culture Collection (ATCC) provided the genomic DNA used to clone Acel_2062 (ATCC Number: ATCC 43068). Protein production and crystallization of the Acel_2062 protein was carried out by standard JCSG protocols
[8 (link)]. Clones were generated using the Polymerase Incomplete Primer Extension (PIPE) cloning method
[9 (link)]. The gene encoding Acel_2062 (GenBank: YP_873820[GenBank:YP_873820]; UniProtKB: A0LWM4[UniProtKB:A0LWM4]) was synthesized with codons optimized for Escherichia coli expression (Codon Devices, Cambridge, MA) and cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino terminus of the full-length protein. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed and dispensed on selective LB-agar plates. The cloning junctions were confirmed by DNA sequencing. Expression was performed in a selenomethionine-containing medium at 37°C. Selenomethionine was incorporated via inhibition of methionine biosynthesis
[10 (link)], which does not require a methionine auxotrophic strain. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 μg/ml, and the cells were harvested and frozen. After one freeze/thaw cycle the cells were homogenized in lysis buffer [50 mM HEPES, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine-HCl (TCEP), pH 8.0] and passed through a Microfluidizer (Microfluidics). The lysate was clarified by centrifugation at 32,500 x g for 30 minutes and loaded onto a nickel-chelating resin (GE Healthcare) pre-equilibrated with lysis buffer, the resin was washed with wash buffer [50 mM HEPES, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0], and the protein was eluted with elution buffer [20 mM HEPES, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0]. The eluate was buffer exchanged with TEV buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] using a PD-10 column (GE Healthcare), and incubated with 1 mg of TEV protease per 15 mg of eluted protein for 2 hours at 20°–25°C followed by overnight at 4°C. The protease-treated eluate was passed over nickel-chelating resin (GE Healthcare) pre-equilibrated with HEPES crystallization buffer [20 mM HEPES, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP, pH 8.0] and the resin was washed with the same buffer. The flow-through and wash fractions were combined and concentrated to 15.6 mg/ml by centrifugal ultrafiltration (Millipore) for crystallization trials.

SOLVE can model raw X-ray data for either MIR or MAD in which the macromolecular structure is defined by a file in PDB format (Bernstein et al., 1977 ▶ ) and heavy-atom parameters are specified by the user. The generate feature allows any degree of ‘experimental’ uncertainty in measurement of intensities. It also allows limited non-isomorphism for MIR data in which cell dimensions differ for native and any of the derivative data sets (but in which the macromolecular structure is identical).
Once a data set has been generated, the SOLVE algorithm then can be applied to the data set in an attempt to solve it. SOLVE can calculate an electron-density map based on the structure input in PDB format and evaluate the correlation coefficient of this map with the maps that it generates during the structure-determination process. For heavy-atom solutions with the inverse hand, this comparison is of course not possible. For heavy-atom solutions which are related to a different origin than the correct solution, the origin shift is automatically determined by SOLVE by finding the origin shift which leads to the closest correspondence of heavy-atom sites in the trial and correct solutions. We use this correlation coefficient as an objective measure of the quality of a heavy-atom solution and as a basis for evaluating the utility of our four scoring criteria.
Model data sets were constructed using the ‘generate’ feature of SOLVE, using two different model proteins. One model protein consisted of coordinates from a dehalogenase enzyme from Rhodococcus species ATCC 55388 (American Type Culture Collection, 1992 ▶ ), determined recently in our laboratory, containing 316 amino-acid residues and crystallizing in space group P2₁2₁2 with cell dimensions a = 94, b = 80, c = 43 Å (J. Newman, personal communication). The other was based on the gene 5 protein structure in space group C2 with cell parameters a = 76, b = 28, c = 42 Å, β = 103° (PDB entry 1bgh; Skinner et al., 1994 ▶ ). For the MIR data ‘experimental’ uncertainties of 3–5% (on intensity) and variation in cell dimensions of 1% from crystal to crystal were used. For the MAD data uncertainties of 2–4% were used. The dehalogenase model was used to generate 132 MIR data sets consisting of a native crystal and two derivative crystals. Each MIR data set contained 6–10 Hg or Au heavy-atom sites with ‘occupancies’ of 0.4–2.6 and thermal factors of 30–50 Ų (although the higher values of ‘occupancy’ are not realistic for this structure, they are included to simulate the effects of a full occupancy Hg or Au in a smaller structure). The gene 5 protein model was used to generate 287 MAD data sets with 4–8 selenomethionine sites with ‘occupancies’ of 0.6–1.4 and thermal factors of 30–50 Ų. All the data sets were generated including anomalous differences. During the course of each structure determination, trial solutions were scored using the four criteria in Table 1 ▶. The Z scores for each trial solution and the correlation coefficients of trial and correct electron-density maps were recorded for all trial solutions which had the correct hand. Those that had the opposite hand were not considered, as our simple correlation-coefficient measure of the actual quality of solutions was not applicable.