Crystallization and structure determination

Crystallization of the purified proteins was initially performed using the following crystal screening kits: Index and PEG/Ion (Hampton Research) and Wizard I and II (Rigaku) using the hanging-drop vapor-diffusion technique at 20 °C. Drop size was 2 μL, which includes 1 μL of protein solution and 1 μL of reservoir solution, and the drop was equilibrated against 50 μL of the reservoir solution. The MsMDH crystals, co-crystallized with NAD⁺ (molar ratio 1:10) to capture the reaction product inside the MsMDH crystal structure, appeared in 16% (w/v) PEG 3350 and 6% (v/v) tacsimate at pH 6.0. The cryoprotectant solution was a mixture of 16% (w/v) PEG 3350, 6% (v/v) tacsimate at pH 6.0, and 30% (v/v) glycerol. Data were collected under 100 K at Beamline 7A of the Pohang Accelerator Laboratory (Pohang, Republic of Korea)⁵³. Subsequently, the data were indexed, integrated, and scaled using the HKL2000 software suite⁵⁴. The MsMDH crystals belonged to a space group P6422 with unit cell parameters of a = 80.09 Å, b = 80.09 Å, and c = 193.15 Å; α = β = 90° and γ = 120°. With one molecule of MsMDH per asymmetric unit, the Matthews coefficient was ~2.58 ų Da⁻¹, which corresponds to a solvent content of ~52.04 %⁵⁵. The structure of MsMDH was determined by molecular replacement with the CCP4 version of MOLREP⁵⁶ using the structure of an MDH from Haemophilus influenzae (PDB code 6BAL, 77% sequence identity) as a search model. The model building was performed using the WinCoot program⁵⁷ and the refinement was performed with REFMAC5⁵⁸. The highest quality CgMDH crystals co-crystallized with l-malate and NAD⁺ (molar ratio 1:20 and 1:10) appeared in 20% (w/v) PEG 3350, 0.1 M HEPES at pH 7.5, and 0.2 M MgCl₂·6H₂O. The cryoprotectant solution includes 20% (w/v) PEG 3350, 0.1 M HEPES at pH 7.5, 0.2 M MgCl₂·6H₂O, and 30% (v/v) glycerol. The CgMDH crystals belonged to the space group C2 with unit cell parameters of a = 102.93 Å, b = 116.94 Å, and c = 66.00 Å; α = γ = 90° and β = 95.31°. Using one molecule of CgMDH per asymmetric unit, the Matthews coefficient was ~2.87 ų Da⁻¹, which corresponds to a solvent content of ~56.77%⁵⁵. The structure of CgMDH was determined by molecular replacement with the CCP4 version of MOLREP using the structure of a MDH from Mycobacterium tuberculosis (PDB code 4TVO, 57% sequence identity) as a search model. The model was built following the procedure described above. Statistical analyses of the data are summarized in Supplementary Table ⁶. The refined models of MsMDH and CgMDH were deposited in the Protein Data Bank with PDB codes 6ITL (10.2210/pdb6ITL/pdb) and 6ITK (10.2210/pdb6ITK/pdb), respectively.