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Proc Natl Acad Sci U S A. Apr 17, 2007; 104(16): 6678–6683.
Published online Apr 10, 2007. doi:  10.1073/pnas.0610337104
PMCID: PMC1850019
Biophysics

Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing

Abstract

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1 sam2 mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 Å by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1 sam2 strain grown in selenomethionine. Six of eight selenium residues were located in the structure.

Keywords: x-ray crystallography, methionine, Saccharomyces cerevisiae

Structural analysis of proteins by x-ray crystallography is a powerful tool to illuminate protein function. Expression of recombinant proteins in Escherichia coli has been used extensively to obtain the large amounts of highly purified protein required for structural analysis. However, many eukaryotic proteins are not soluble in E. coli (1), and many eukaryotic proteins bear posttranslational modifications that are not added in E. coli because of the lack of requisite modification enzymes. Thus, expression of many proteins may require a eukaryotic host.

The yeast Saccharomyces cerevisiae, which is simple and inexpensive to culture, is attractive as a eukaryotic expression host. High-level protein expression and affinity purification, yielding 2 mg of purified protein per liter of culture (2, 3), has been demonstrated in appropriate strains. The use of S. cerevisiae as both a homologous and heterologous expression system for x-ray crystallography is established. The structures of a number of proteins have been determined with protein expressed in yeast, including yeast Rad6 (4), and Ade1 (5), as well as the catalytic domain of human ADAR2 (6) and the rabbit Ca2+-ATPase (7), a membrane protein.

A major impediment to using S. cerevisiae as a routine expression host for x-ray crystallography has been the inability to efficiently replace methionine residues with selenomethionine for anomalous dispersion phasing. In contrast to either multiple isomorphous replacement or molecular replacement (8), multiwavelength anomalous dispersion (MAD) and single-wavelength anomalous dispersion (SAD) phasing of selenomethionine-containing crystals provide a solution to the problem of phase determination that can be applied to many proteins (9, 10). The structural and functional identity of methionine and selenomethionine derivatives is well established because selenomethionine replacement is easily effected in E. coli. Furthermore, the use of MAD and SAD phasing has increased with advances in synchrotron beam lines and automated methods for structure solution, such as SOLVE (11), RESOLVE (12), SnB (13), and BnP (14), to locate the anomalous scattering atoms.

Efficient replacement of methionine with selenomethionine is much more difficult in organisms other than E. coli, in which nearly 100% replacement is routine. The problem with selenomethionine incorporation in S. cerevisiae is likely due to the toxicity of selenomethionine because growth ceases within minutes after addition of 10 μg/ml selenomethionine (15). Previous attempts to obtain selenomethionine resistant strains in S. cerevisiae have not been helpful either for elucidating the basis of toxicity or for improving incorporation of selenomethionine into proteins. Selenomethionine resistant mutants that have been isolated either increase the intracellular methionine pools or block the uptake of selenomethionine (16).

We considered it likely that selenomethionine is fully competent to replace methionine in protein synthesis. Investigations in the 1970s had suggested that selenomethionine competed efficiently with methionine in charging tRNA (17) and in incorporation into proteins in vivo (15). Furthermore, we thought it unlikely that replacement of a particular methionine with selenomethionine in an essential protein is the primary cause of its toxicity, because Singer et al. (15) found that RNA synthesis is inhibited within minutes after addition of selenomethionine, whereas protein synthesis continues for >40 min. Some recent attempts to incorporate selenomethionine in yeasts have met with limited success, but in most cases have resulted in only partial substitution. Bushnell et al. (18) obtained ≈50% selenomethionine incorporation into RNA polymerase II, which was insufficient for phasing, but the selenomethionine peaks did serve as markers to guide model building. Larsson et al. (19) reported 40–50% selenomethionine incorporation for two proteins expressed in Pichia pastoris, in one case, the selenomethionine was used for MAD phasing. Laurila et al. (20) obtained fully selenomethionyl substituted RNA-dependent RNA polymerase in S. cerevisiae, but induction of expression and selenomethionine incorporation occurred in the absence of cell growth, in which many proteins are poorly expressed.

We hypothesized that the toxicity of selenomethionine is due to its conversion to the seleno derivative of S-adenosylmethionine (Se-AdoMet). Either Se-AdoMet itself or one of its metabolic products could be toxic. S-adenosylmethionine (AdoMet), the primary metabolic product of methionine, is involved in a number of important processes, including methylation of proteins, RNAs, and lipids (21) and the biosynthesis of biotin (22) and polyamines (Fig. 1A) (23, 24). It has been estimated that only ATP participates in more reactions than AdoMet (21). In addition, a product of AdoMet metabolism, such as S-adenosylhomocysteine, homocysteine, cysteine, or glutathione (21), could have toxic effects as selenium derivatives; for example, S-adenosylhomocysteine, itself a potent inhibitor of many methyltransferases (21), could be more inhibitory as a seleno derivative. Furthermore, there are two AdoMet synthetases in yeast (encoded by SAM1 and SAM2), either of which suffices for synthesis of AdoMet. Thus, one would not be able to obtain an AdoMet mutant in a simple genetic screen for selenomethionine resistance, if Se-AdoMet were the cause of the toxicity. Moreover, because AdoMet is required for life, a sam1 sam2 mutant would only grow if AdoMet was supplied in the medium (21).

Fig. 1.
Selenomethionine toxicity and S-adenosylmethionine synthesis. (A) Diagram of methionine and AdoMet metabolic pathways in S. cerevisiae. (B) Selenomethionine resistance of sam1Δ and sam2Δ strains. Yeast strains bearing the indicated mutations ...

We report here that a mutant strain of S. cerevisiae deleted for both SAM genes exhibits significantly enhanced resistance to selenomethionine, and allows efficient incorporation of selenomethionine into proteins. Replacement of methionine with selenomethionine is nearly complete based on quantitative mass spectrometry, and permitted structural determination of S. cerevisiae tryptophanyl-tRNA synthetase, Wrs1 protein, using MAD phasing techniques. Thus, this manipulation of metabolism provides a general solution to the problem of selenomethionine incorporation in S. cerevisiae.

Results

Selenomethionine Resistance Is Conferred by Blocking Conversion of Methionine to S-Adenosylmethionine.

If Se-AdoMet, or one of its metabolites, is the primary toxic compound inhibiting growth of yeast cells in selenomethionine, then preventing conversion of selenomethionine to Se-AdoMet should allow yeast to grow on selenomethionine. Because two highly homologous yeast genes (SAM1 and SAM2) each encode an AdoMet synthetase, the enzyme responsible for this reaction (21, 25), we created a double mutant strain in which both of these genes were precisely deleted, and replaced by drug resistance cassettes derived from the yeast knockout collection (26). The resulting sam1Δ sam2Δ double mutant strain should block conversion of selenomethionine to Se-AdoMet (see Fig. 1A) and require AdoMet for growth (21).

As shown in Fig. 1B, the sam1Δ sam2Δ double mutant strain grows well on media containing as much as 120 μM selenomethionine, whereas growth of the wild-type strain is severely inhibited on media containing as little as 30 μM selenomethionine. Both single mutants have intermediate effects; the sam2Δ mutant is slightly more resistant to 30 μM selenomethionine than the wild-type strain, whereas the sam1Δ mutant is nearly as resistant as the double mutant strain at 60 μM selenomethionine but sensitive at 120 μM. Resistance to selenomethionine was also observed in a sam1Δ sam2Δ mutant created in a different strain background (data not shown).

There are two possible explanations for the selenomethionine resistance of the mutants blocked in AdoMet synthesis: either failure to convert selenomethionine to Se-AdoMet could result in the absence of the toxic compound, or failure to consume methionine in AdoMet synthesis could result in an increase in the intracellular methionine concentration, which in turn competes with selenomethionine (see Fig. 1A). To distinguish between these possibilities, we tested the importance of the intracellular methionine pool in selenomethionine resistance by deleting the met6 gene, which blocks the conversion of homocysteine to methionine (Fig. 1A). We find that the sam1Δ sam2Δ met6Δ triple mutant grows better on selenomethionine compared with the SAM1+SAM2+met6Δ strain (Fig. 1C). Thus we infer that the selenomethionine resistance of the sam1Δ sam2Δ double mutant is primarily due to blocking conversion of selenomethionine to a toxic compound.

sam1Δ sam2Δ Mutants Efficiently Produce Protein in the Presence of Selenomethionine.

To assess the usefulness of the selenomethionine resistant mutant strains for the preparation of selenomethionine-substituted proteins, we examined the yield of ORF fusion proteins expressed in the wild-type and mutant strains during growth in selenomethionine. We first generated a plasmid for expression of ORFs (under control of the GAL1 promoter) that were fused at their C terminus to a tripartite tag containing a site recognized by 3C protease, His6, an HA epitope and the ZZ domain of protein A, similar to that used in the MORF collection (2). Protein expression was induced by addition of galactose with or without simultaneous addition of selenomethionine, and the amount of protein produced was assessed after affinity purification on IgG resin and SDS/PAGE.

The amount of Ncl1-fusion protein expressed after 24 h induction in either 0.1 mM or 0.5 mM selenomethionine was substantially greater in the sam1Δ sam2Δ strain than in the wild-type strain (Fig. 2A, compare lanes h and i with d and e). The sam1Δ sam2Δ mutant also grew better than the wild-type strain in both conditions, based on OD600. In media lacking selenomethionine, Ncl1 protein is produced in good yield in all strains except the sam1Δ sam2Δ met6Δ triple mutant (Fig. 2 and data not shown).

Fig. 2.
Evaluation of protein expression in sam1Δ sam2Δ and sam1Δ met6Δ mutants during growth in media containing selenomethionine. (A) Analysis of Ncl1 protein expression. Ncl1-fusion protein was induced with galactose in the ...

Analysis of Wrs1 expression confirms that growth (data not shown) and protein production in selenomethionine are significantly better in the sam1Δ sam2Δ mutant compared with the wild-type strain (Fig. 2B). Examination of five other proteins (Rad6p, Dps1p, Dus1p, Mes1p and Gus1p) confirms that this method is generally applicable for expression in selenomethionine. In each case, we observed efficient protein production in the sam1Δ sam2Δ mutant in selenomethionine at ≈30–60% of the amount obtained from cells grown in sulfate (data not shown). We note that there are likely some protein-specific differences in expression in selenomethionine in that addition of 0.5 mM selenomethionine has a more inhibitory effect on Wrs1 protein production relative to that observed with Ncl1.

We also examined protein expression in the sam1Δ SAM2+ met6Δ strain after transient repression of SAM2 by addition of 1 mM choline and 50 μM myoinositol (27, 28), coincident with induction of ORF fusion expression. Incorporation of selenomethionine could in principle be more efficient in a met6Δ background which should contain reduced levels of intracellular methionine. However, we find that production of proteins in 0.2 mM selenomethionine is poor in this strain and is only marginally better in conditions in which SAM2 is repressed (Fig. 2). Thus, despite the fact that this strain is quite resistant to selenomethionine (Fig. 1C), it is not useful for protein production in these conditions.

Selenomethionine Incorporation Is Nearly Complete, Based on Mass Spectrometry.

The usefulness of protein purified from the sam1Δ sam2Δ mutant for MAD/SAD phasing depends primarily upon the fraction of methionine that is replaced by selenomethionine. To judge the efficiency of selenomethionine substitution, we used quantitative mass spectrometry, based on differential isotope labeling of two samples, to measure the specific reduction in methionine-containing tryptic peptides in Ncl1 protein that was prepared from yeast grown in selenomethionine. Differential isotope labeling of Ncl1 protein (Fig. 3A) was accomplished by digestion with trypsin in either H216O or H218O (29, 30); tryptic peptides will then be labeled at their newly generated C termini with one or two 18O (16O) molecules, resulting in a m/z difference of 2 or 4 (31). Mixtures of H216O trypsin-digested and H218O trypsin-digested Ncl1 protein were cospotted and analyzed by MALDI-TOF, allowing direct comparison of signal intensities of chemically identical but differentially labeled peptides (29). We first identified 6 easily detectable tryptic peptides derived from Ncl1 protein. We established that the + 2 and + 4 signals from these peptides can be attributed solely to the Ncl1 from the 18O sample, and that the protein digested in 18O contributes little (if any) signal to the 16O bands (data not shown). Four of these peptides do not contain methionine and are expected to exhibit equal representation in Ncl1 protein whether or not the media contains selenomethionine; two peptides contain methionine and are expected to show reduced representation if their methionine is replaced by selenomethionine.

Fig. 3.
Quantification of individual peptides from purified Ncl1 after growth in selenomethionine. (A) SDS/PAGE of purified Ncl1 protein. Lanes: a, Bio-Rad markers; b, 15 μg of Ncl1 protein from culture grown in sulfate; c–g, 3 μg of Ncl1 ...

As shown in Fig. 3B, the two methionine-containing peptides are severely depleted in Ncl1 protein derived from yeast grown in 0.5 mM selenomethionine compared with Ncl1 protein from media lacking selenomethionine. The mixture of equal amounts of trypsin-digested Ncl1 protein made from cultures grown in sulfate and in 0.5 mM selenomethionine yields similar signals for the four peptides that do not contain methionine (Fig. 3B). In each case, there is slightly more signal for the non-methionine containing peptide from the selenomethionine-grown sample than from the corresponding peptide from the sulfate grown sample (which is set at 100%). By contrast, there is dramatically less signal (2% and 5% respectively) for the two methionine-containing peptides from the selenomethionine-grown sample compared with the corresponding peptides from the sulfate-grown sample. This result demonstrates that >95% of the methionine is replaced by selenomethionine in the Ncl1 protein grown in 0.5 mM selenomethionine. For one of these peptides (LNSANLMVVNHDAQFFPR of m/z 2073.31), we also observe an increase in the corresponding selenomethionine-containing peptide (Fig. 3C) with increasing selenomethionine in the media, although this peptide cannot be quantified relative to the corresponding methionine peptide because they are chemically different.

We also examined the effect of selenomethionine concentration in the media on the efficiency of selenomethionine incorporation. We observed 83% incorporation at 0.125 mM selenomethionine, 90% at 0.25 mM, and 95% at 0.5 mM (Fig. 3C).

Structure of Yeast Wrs1.

To further establish that the sam1Δ sam2Δ S. cerevisiae strain efficiently incorporates selenomethionine into proteins, we determined the crystal structure of S. cerevisiae Wrs1 protein (eight Met residues), produced in this strain, using MAD phasing techniques. Selenium incorporation was verified based on fluorescence scans at the beamline and by the anomalous difference electron density shown in Fig. 4. The selenium substructure of Wrs1 was solved by using BnP (14), with the positions of six of the eight Se atoms identified in the top 8 peaks, producing phases with an initial mean figure of merit of 0.57. Subsequent phase refinement in BnP and RESOLVE (32) resulted in a mean figure of merit increase to 0.78. Fourier maps generated at this stage were easily interpretable, allowing for facile building of the initial protein model (Fig. 4).

Fig. 4.
Representative electron density for yeast Wrs1. A MAD experimental electron density map, calculated at 3.0 Å and contoured at 1σ (blue), is shown for a region containing Met-169, Met-174, and Met-360 of yeast Wrs1. The map was generated ...

The final model of yeast Wrs1 consists of 371 residues, corresponding to residues 99–477 based on sequence alignment to the human enzyme (33). The N-terminal 46 residues (residues 54 through 98) and the region containing the conserved KMSKS motif (residues 345–352) are disordered and not included in the model. SDS/PAGE analysis of dissolved crystals results in a single band of protein migrating at a molecular mass of ≈45 kDa, indicating a loss of ≈5 kDa from the theoretically calculated molecular mass (factoring in the additional 8 residues left at the C terminus after 3C protease cleavage) and consistent with the loss of the first ≈45 residues of the protein (data not shown). Hence, the structure of Wrs1 reported here represents the catalytic fragment of full length Wrs1, generated through proteolysis of the N terminus during purification and similar to that reported for human Wrs1 protein (33). Taken together, these observations explain why only six of the possible eight selenium sites were located by BnP. Analysis of an anomalous difference electron density map, calculated by using the anomalous differences from the peak data set and the phases derived from BnP and RESOLVE clearly define the positions of the selenium atoms for the six ordered methionine residues in the protein model (Fig. 4). Detailed analysis of Wrs1 will be reported elsewhere.

Discussion

We have demonstrated that preventing the conversion of selenomethionine to Se-AdoMet dramatically reduces the toxicity of selenomethionine in S. cerevisiae, allowing both better growth in the presence of selenomethionine and nearly complete replacement of methionine with selenomethionine in expressed protein. Based on quantitative mass spectrometry, >90% of the methionine is replaced by selenomethionine in the presence of 0.25–0.5 mM selenomethionine. These growth conditions are likely to be generally applicable for incorporation of selenomethionine because we have produced seven proteins in the sam1Δ sam2Δ mutant grown in selenomethionine. Furthermore, the structure of Wrs1 protein was solved by using MAD phasing of protein purified from the sam1Δ sam2Δ mutant grown in selenomethionine. Thus it is now feasible to consider yeast as a source of proteins for structural genomics, because phasing issues are greatly eased by efficient selenomethionine incorporation. We speculate that these results may provide a general solution for production of selenomethionine containing proteins in other organisms.

Methods

Strain Construction and Growth.

Yeast strains [supporting information (SI) Table 2] derived from strain Y258 (MATa, pep4-3, his4-580, ura3-52, leu2-3, 112) (2) were constructed by transformation with kanMX and nat1 cassettes as described (26, 34, 35). Oligonucleotides for amplification and confirmation are listed in SI Table 3.

Selenomethionine resistance was assayed by growth of cultures overnight in YPD (with 60 μM AdoMet as required), followed by dilution in YPD to OD600 = 0.5, growth for 4 h, dilution to OD600 = 1.25, spotting 2.5 μl of 5-fold serial dilutions on synthetic (S) dropout media (lacking methionine or complete as indicated) containing 60 μM AdoMet and selenomethionine as indicated, and incubation at 30°C.

Protein Affinity Purification.

ORFs were cloned under PGAL control into vector BG2483, a 2μ URA3 vector derived from BG1766 (2), which expresses ORFs fused at their C terminus to a multipart tag (3C site-HA epitope-His6-ZZ domain of protein A) and which has been modified for ligation independent cloning. Yeast transformants were grown overnight in SD lacking uracil containing 64 μM AdoMet, diluted 20-fold in S dropout media lacking uracil and methionine, containing 2% raffinose (36) and 64 μM AdoMet, and grown for 8 h at 30°C, followed by dilution to OD600 of 0.025, and overnight growth. At OD600 ≈1, cultures were induced by addition of galactose to 2% (and selenomethionine and other additions, as indicated), and growth was continued for 24 h, followed by harvesting of cells. Expressed protein was evaluated from crude extracts by binding to IgG Sepharose (2), elution of bound protein by boiling the beads in loading buffer, and SDS/PAGE. Proteins subjected to mass spectrometry were removed from IgG Sepharose by 3C protease cleavage (2), subjected to SDS/PAGE, and eluted as described (37). Purification of Wrs1 protein for crystallization involved growth of 18 liters of culture, IgG Sepharose chromatography, 3C protease cleavage, and gel filtration on a HiLoad 16-60 Superdex 200 prep grade (Amersham, Piscataway, NJ) column.

Quantitative MALDI Mass Spectrometry.

Samples were prepared for mass spectrometry analysis by mixing two portions of tryptic peptides extracted from gel samples and digested as described (37), one from the H216O and the other from 95% H218O (Isotec) digestions. MALDI spectra, obtained on a Voyager DE STR MALDI-TOF instrument (Applied Biosystems, Framingham, MA), were acquired in positive ion reflectron mode as described (38), with an accelerating voltage of 20 kV and a 150-ns delay time. Spectra were obtained by averaging 200 acquisitions from single laser pulse (20 Hz, Voyager, N2 337-nm laser). Proteins were identified by using ion searches performed on the processed spectra against the NCBI NR database using the MASCOT search engine (Matrix Science, Inc., Boston, MA) with carbamidomethyl cysteine and oxidized methionine as variable modifications (www.ebi.ac.uk/IPI/IPIhuman.html), to determine possible sequence correlations of known proteins with variable carbamidomethyl cysteine, oxidized methionine and selenomethionine modifications.

Crystallization and Data Collection.

Wrs1 [4.8 mg/ml in 20 mM Hepes (pH 7.5), 5% (vol/vol) glycerol, 500 mM sodium chloride, 2 mM DTT, 0.025% (wt/vol) sodium azide] was screened for crystallization by using a high throughput microbatch-under oil technique (39). After 1 week at 4°C, 19 of 1,536 cocktails produced conditions suitable for optimization. One mixture [80% (vol/vol) polyethylene glycol 400 (PEG 400), 100 mM ammonium bromide, 100 mM Mops (pH 7.0)] was optimized for crystallization by volumetric codilution of the protein and mixture solutions, using a modified version of a batch protocol (40). Crystals of the Se-Met derivative of Wrs1 (5.0 mg/ml) were produced by using the same technique.

Crystals of the native and Se-Met derivative of Wrs1 were flash frozen in liquid nitrogen using the high concentration of PEG 400 from the crystallization mixture as the cryoprotectant. Multiwavelength anomalous x-ray diffraction data to 3.0 Å were collected at beamline 9-1 at the Stanford Synchrotron Research Laboratory (SSRL) by using remote data collection techniques (41, 43, **). Data sets were measured at the selenium K-edge peak (0.97935 Å), inflection point (0.97952 Å), and remote (0.96109 Å) energy wavelengths. Native crystals of Wrs1, which diffracted to 1.8 Å, were also collected remotely by using beamline 11-1 at SSRL. All data were processed by using MOSFLM (44) combined with SCALA from the CCP4 suite of programs (45). The data collection statistics are summarized in Table 1.

Table 1.
Data collection, phasing, and refinement statistics for Wrs1p

Structure Solution and Refinement.

The structure of Wrs1 was solved by using MAD phasing techniques. The heavy-atom substructure was solved by using BnP (14). Wrs1 protein was crystallized in the tetragonal space group P4x212. As such, the enantiomorph determination feature of BnP was used to determine the correct space group. By using unrefined protein phases, the ratio of the standard deviations of the electron density in the protein and solvent regions was 1.11 for P41212 and 1.81 for P43212, indicating that the latter was the true space group. The refined phases from BnP were input into RESOLVE (32) for density modification and automated chain tracing. RESOLVE was able to position 196 of 432 residues. Fourier maps were generated from the refined and solvent-flattened phases output from RESOLVE and used to adjust the initial protein model and manually build an additional 175 residues into the electron density using COOT (46).

The model was initially refined with CNS (47). Rigid body refinement, followed by cycles of positional and group B factor refinement (main chain/side chain) resulted in R and free R values of 27.1% and 31.1%, respectively. Iterative cycles of model building in COOT followed by positional and group B factor refinement were carried out to adjust flexible loop regions and build side chain residues. The resulting atomic model was then used as a starting point for refinement of the isomorphous high-resolution data set. Iterative cycles of refinement (positional and individual B factor refinement) followed by model building in COOT dropped the R and free R factors to 25.4% and 28.0%, respectively. Strong electron density was observed in the 2FoFc and FoFc electron density maps calculated at this stage for four PEG 400 molecules, which were built in. A total of 358 water molecules were also added. In the final stages of refinement, the TLS feature of REFMAC 5.0 (42, 48) was applied. Residues 99–153 (N-terminal), 154–362 and 453–471 (catalytic), and 363–452 (C-terminal) were defined as distinct domains with TLS to model anisotropic displacements of each domain as a rigid-body. Final refinement statistics are summarized in Table 1.

Supplementary Material

Supporting Tables:

Acknowledgments

We thank M. Dumont for advice and comments and Dr. Eddie Snell for advice and assistance during remote data collections at the Stanford Synchrotion Radiation Laboratory (SSRL). This work was supported by National Institutes of Health (NIH) Grant NIH 1 U54 GM074899 establishing the Center for High Throughput Structural Biology. Portions of this research were carried out at SSRL, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research and by the NIH, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.

Abbreviations

AdoMet
S-adenosylmethionine
Se-AdoMet
Se-adenosylselenomethionine
MAD
multiwavelength anomalous dispersion
SAD
single-wavelength anomalous dispersion.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Data deposition: The crystallographic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2IP1).

This article contains supporting information online at www.pnas.org/cgi/content/full/0610337104/DC1.

**Gonzáles, A., Moorhead, P., McPhillips, S., Sauter, N. K. (2005) Acta Crystallogr A 61:C486 (abstr. P.25.03.1).

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