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Science 24 May 2002:
Vol. 296. no. 5572, pp. 1462 - 1466
DOI: 10.1126/science.1069556

A New UAG-Encoded Residue in the Structure of a Methanogen Methyltransferase
Bing Hao, Weimin Gong, Tsuneo K. Ferguson, Carey M. James, Joseph A. Krzycki, and Michael K. Chan

Supplementary Material

Materials and Methods

Crystallization and data collection. MtmB was isolated as described previously (S1) with the exception that the procedure was performed aerobically. Under these conditions, MtmB forms a relatively stable complex with MtmC. Crystallization attempts on the MtmBC complex were performed at 4°C using the hanging-drop vapor-diffusion method (S2). Drops contained equal volumes of reservoir and protein solution (30 mg/ml protein, 200 mM NaCl, and 50 mM MOPS, pH 6.0). Clear crystals of similar shape could be obtained from several different crystallization conditions, and two of these were used in the structural analysis [form 1: 4.3 M NaCl, and 0.1 M Hepes (pH 7.5); and form 2: 1.6 M (NH₄)₂SO₄, 0.1 M NaCl and 0.1 M Hepes (pH 7.5)]. SDS-PAGE gels of washed and redissolved crystals revealed that they contained only MtmB. Before data collection, the crystals were transferred gradually into the reservoir solution containing increasing amounts of glycerol up to 30% (v/v). The two high-resolution native data sets were collected at the BIOCARS sector 14C of the Advance Photon Source (APS) at Argonne National Laboratory. The data for the heavy atom derivatives were collected at beamline 9-2 at Stanford Synchrotron Radiation Laboratory (SSRL), BIOCARS sector 14D at APS, and beamline X4A at the National Synchrotron Light Source in Brookhaven National Lab. For each heavy-atom derivative, anomalous diffraction data were collected at the wavelength corresponding to the peak anomalous scattering, except for iodine- and cesium-containing derivatives, which were collected at low energy to optimize the anomalous signal. Data processing and reduction were performed using DENZO and SCALEPACK (S3).

Phase determination and refinement. The structure of MtmB was determined by the multiple isomorphous replacement and anomalous scattering (MIRAS) method. More than 200 crystals were treated with heavy atom compounds, and a total of about 60 data sets were collected but only 6 data sets were used in final phase calculation. The Hg and Pb sites were initially located by isomorphous and anomalous difference Patterson maps independently. All the other derivatives were solved by difference Fourier using the Hg/Pb phases. The best derivative was obtained with sodium iodide (Table 1). Initially heavy atom parameters were refined and MIRAS phases were calculated and compared by using PHASES (S4) and MLPHARE (S5) from CCP4 (S6). These phases were further improved by solvent flattering and histogram matching with DM (S7) and maximum likelihood density modification with RESOLVE (S8, S9). Model building was carried out with the program O (S10). Despite the weak anomalous signals and common sites, at this stage, over 300 residues of poly(Ala) model could be built into this map. Phases calculated from this preliminary model were recombined with the experimental phases using PHASES (S4), CNS (S11), and SIGMAA (S12), respectively. The density map calculated from the recombined phase was used to adjust and complete the backbone model. The secondary structure prediction from the MtmB amino acid sequence by GOR IV (S13) was used to confirm the correctness of the trace. Multiple cycles of such model building and phase improvement were carried out until over 90% of visible side chains had been built. Structure refinement was performed using CNS (S11) with 10% of the data omitted for the Free-R factor calculation. No sigma cutoff was used. Secondary structure assignments: Greek Letter Alpha helixes: 1, A9-A18; 2, A24-A42; 3, A56-A73; 4, A88-A95; 5, A137-A148; 6, A174-A193; 7, A210-A214; 8, A240-A251; 9, A271-A288; 10, A309-A325; 11, A343-A359; 12, A380-A393; 13, A398-A412; 14, A438-A453; Greek Letter Beta strands: 1, A19-A21; 2, A75-A77; 3, A82-A87; 4, A101-A104; 5, A111-A114; 6, A125-A135; 7, A155-A161; 8, A201-A203; 9, A227-A231; 10, A255-A264; 11, A293-A300; 12, A330-A336; 13, A363-A368; 14, A428-A430; 15, A434-A436.

Supplemental Figure 1. Structure and folding of an MtmB subunit. (A) Ribbon diagram of one MtmB subunit. The secondary structure elements are color-coded with Greek Letter Alpha helices as blue, Greek Letter Beta sheets as red, and random coils as green. The atoms of the UAG-encoded residue are shown as ball-and-stick models and are colored by their elements, with carbon as gray, nitrogen as blue, and oxygen as red. (B) Electrostatic potential energy surface diagram of MtmB in a similar orientation. (C) Stereoview of C Greek Letter Alpha -trace in rainbow from blue (NH₂-terminus) to red (COOH-terminus) with every 20th amino acid labeled.

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Supplemental Figure 2. Electron density maps of the NaCl crystal form. (A) 2F_O - F_C omit map at 3 Greek Letter Sigma for the 4-methyl-pyrroline carboxylate group of the UAG-encoded residue for crystals grown in NaCl solution with the two ring orientations used to fit it. The carbons of orientation 1 are colored in pink. In orientation 2 they are colored in tan. (B) F_O - F_C difference map at 4.5 Greek Letter Sigma for NaCl crystals after incorporation of model for orientation 2 at 15% occupancy. (C) Fit of orientation 1 model to this density. (D) F_O - F_C difference map at 4.5 for NaCl crystals after incorporation of model for orientation 1 at 85% occupancy. (E) Fit of orientation 2 model to this density.

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Supplemental Figure 3. Electron density maps of the (NH₄)₂SO₄ crystal form. (A) 2F_O - F_C omit map at 3 Greek Letter Sigma for the 4-methyl-pyrroline carboxylate group of the UAG-encoded residue for crystals grown in high (NH₄)₂SO₄ solution with the two ring orientations used to fit it. The carbons of orientation 1 are colored in pink. In orientation 2 they are colored in tan. (B) F_O - F_C difference map at 4.5 Greek Letter Sigma for (NH₄)₂SO₄ crystals after incorporation of model for orientation 2 at 60% occupancy. (C) Fit of orientation 1 model to this density with additional ammonium ion. (D) F_O - F_C difference map at 4.5 for (NH₄)₂SO₄ crystals after incorporation of model for orientation 1 at 60% occupancy. (E) Fit of orientation 2 model to this density with additional amine group added to the pyrroline ring.