(a) Domain organization of protein MJ0100. (b) Sequence alignment of the CBS-domain pair of MJ0100 and related proteins. The figure shows a ClustalW (Larkin et al., 2007 ▶) alignment of the C-terminal part of some of the proteins that contain a combination of DUF39 and CBS domains. The number in brackets indicates the number in the protein sequence of the first amino acid in the alignment. The beginning of the MJ0100c fragment is shown by a triangle. Colour codes: red on yellow background, invariant amino acids; black on blue, strongly conserved; black on green, similar; green on white, weakly similar; black on white, not conserved. The proteins shown and the accession numbers of the proteins in the alignment are MJ0100 from Methanococcus jannaschii (NP_247064), MM_0241 from Methanosarcina mazei Gö1 (AAM29937), DUF39 from Methanococcoides burtonii (YP_565063), RCIX1241 from uncultured archaeon RC-I (YP_685869), DUF39 from uncultured archaeon GZfos26B2 (AAU83016), AF1186 from Archeoglobus fulgidus DSM 4304 (NP_070015), MMP1359 from Methanococcus maripaludis S2 (NP_988479), DUF39 from Methanococcus vannielii SB (YP_001323187), DUF39 from Methanococcus aeolicus Nankai-3 (YP_001324988), DUF39 from Methanothermobacter thermautotophicus strain Delta H (NP_275992), DUF39 from Methanopyrus kandlrei AV19 (NP_614190), Mlab_0451 from Methanocorpusculum labreanum Z (YP_001029892), DUF39 from Methanoculleus marisnigri Jr1 (YP_001047428), DUF39 from Methanospirillum hungatei JF-1 (YP_501633) and DUF39 from Methanosaeta thermophila PT (YP_842462).

(a) Crystal of MJ0100c grown in 15% PEG 10 000, 100 mM Tris–HCl pH 8.8. (b) Crystal of SeMet-labelled MJ0100c grown in 10% PEG 8000, 100 mM Tris–HCl pH 8.6, 1 mM DTT. Crystal size was 0.3 mm.

κ = 180° section of the self-rotation function of the native MJ0100c data set. CBS-domain pairs usually dimerize in a head-to-head or a head-to-tail manner, both being related by pseudo-twofold symmetry. As shown here, there are two noncrystallographic twofold axis (marked as 1 and 2) in the yz plane that would be consistent with the presence of two dimers (four molecules) in the asymmetric unit. However, the V _M (7.29 ų Da⁻¹) and solvent content (83.14%) values would be too high in this case. Consequently, an additional twofold NCS axis parallel to any of the crystallographic axes a, b or c may be present (although it cannot be distinguished because it overlaps with the crystallographic axis). Consequently, eight molecules (four dimers) in the asymmetric unit may be the most probable value. No additional peaks are observed in the κ = 60°, 90° or 120° sections of the self-rotation function. This figure was prepared using MOLREP (Vagin & Teplyakov, 1998 ▶).

Purification of protein MJ0100c. SDS–PAGE analysis of fractions obtained in a standard purification scheme, as reported in §2. Lane 1, molecular-weight markers (in kDa); lane 2, BL21-Codon Plus (DE3) + pML1 before induction; lane 3, BL21-Codon Plus (DE3) + pML1 after 3 h induction with IPTG; lane 4, soluble lysate after ultracentrifugation; lane 5, 20 µg sample after phosphocellulose chromatography; lane 6, 20 µg sample after MonoS chromatography; lane 7, 20 µg sample after gel-filtration chromatography.

Representative X-ray diffraction image from (a) native and (b) SeMet-labelled MJ0100c crystals. The diffraction ring observed in (a) at around 7–8 Å is caused by the presence of silicone oil, which was used as a cryoprotectant in freezing the native crystals. The size of the native and derivatized crystals were 0.4 and 0.3 mm, respectively, and they were exposed for 10 s over a 1° oscillation range. The edge of the detector corresponds to a resolution of 2.13 Å in (a) and 3.3 Å in (b).