2.  Materials and methods  

2.1.  Protein expression and purification   
The sequence encoding the N-terminal domain of hMLH1 (residues 1–340) was amplified by PCR and subcloned into the pET-28-MHL vector (GenBank deposition ID EF456735) downstream of the polyhistidine affinity tag. The protein was overexpressed in Escherichia coli BL21 (DE3) V2R-pRARE cells in Terrific Broth medium in the presence of 50 µg ml−1 kanamycin. The cells were grown at 37°C to an OD600 nm of 1.5, induced by the addition of 1 mM isopropyl β-d-1-thio­galactopyranoside (IPTG) and incubated overnight at 15°C. The cells were harvested by centrifugation at 7000 rev min−1 and resuspended in 50 mM HEPES pH 7.4, 500 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS, 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by passage through a microfluidizer (Microfluidics Corporation) at 138 MPa. After clarification of the crude extract by high-speed centrifugation, the lysate was applied onto a 5 ml HiTrap Chelating column (GE Healthcare) charged with Ni2+. The column was washed with ten column volumes of 20 mM HEPES pH 7.4 containing 500 mM NaCl, 50 mM imidazole and 5% glycerol. The protein was eluted in 20 mM HEPES pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol and then loaded onto a Superdex 200 (26/60, GE Healthcare) column equilibrated in 20 mM PIPES pH 6.5 buffer containing 250 mM NaCl. TEV protease was added to the combined fractions containing MLH1. The protein was further purified to homogeneity by ion-exchange chromatography on a Source 30S column (10/10; GE Healthcare) and eluted in a final buffer consisting of 20 mM PIPES pH 6.5, 250 mM NaCl.

2.2.  Crystallization and structure determination   
Purified MLH1 protein (10 mg ml−1) was mixed with ADP at a 1:5 molar ratio of protein:ligand and crystallized using the sitting-drop vapor-diffusion method by mixing 1 µl protein solution with 1 µl reservoir solution consisting of 20% PEG 4000, 10% 2-propanol, 0.1 M HEPES pH 7.5.
Diffraction data were collected on beamline 19ID at the Advanced Photon Source, Argonne National Laboratory. Reflection intensities from 150 1° diffraction images were initially integrated and scaled using HKL-3000 (Minor et al., 2006 ▸). Using the crystal structure of E. coli MutL (PDB entry 1b62; 36% amino-acid sequence identity; Ban et al., 1999 ▸; Johnson et al., 2008 ▸) as the search model, the structure was solved by molecular replacement with MOLREP (Vagin & Teplyakov, 2010 ▸). The initial refinement alternated cycles of restrained refinement including TLS parameterization in REFMAC (Murshudov et al., 2011 ▸; Winn et al., 2001 ▸) with interactive rebuilding in Coot (Emsley et al., 2010 ▸). After renewed processing of the same diffraction images with XDS (Kabsch, 2010 ▸) and additional scaling with AIMLESS (Evans & Murshudov, 2013 ▸), the model was further refined using autoBUSTER (Blanc et al., 2004 ▸; Bricogne et al., 2011 ▸) and REFMAC interspersed with interactive rebuilding.
The MolProbity statistics of the model compared favorably with a set of reference structures with similar data resolution (MolProbity server v.4.1-537). The model was deposited in the PDB using the PDB_EXTRACT tool (Yang et al., 2004 ▸) with accession code 4p7a. Data-collection, model-refinement and validation statistics are summarized in Table 1 ▸. All figures were prepared using PyMOL (v.1.5.0.4; Schrödinger).