Extended Data Fig. 3: Crystallization of ECOR31 mCpol and mechanism of adenine base specificity.
From: A miniature CRISPR–Cas10 enzyme confers immunity by inhibitory signalling
(a) Size exclusion chromatography (SEC) of ECOR31 mCpol with ENLYFQ cleavage site (red arrow) versus a molecular weight standard. MW of standard peaks in kD: 670, 158, 44, 17, 1.35). (b) Determination of mCpol multimerization state by comparison with a molecular weight standard. The peak at ~48 mL represents the void volume of the column and the peak at ~83 mL (red arrow) is a dimer of mCpol. (c) Purification of mCpol for crystallography (trays set up with post-SEC fraction, red arrow). (d) Conservation of residues governing base specificity in an alignment of 163 mCpol domain-containing proteins. Alignment position 35 equates to S30 and position 39 equates to N34 in ECOR31 mCpol. (e) Single crystal resulting from the 0.1 M bicine, pH 8.0, 15% PEG 1500 condition, mixed 1:1 with protein and supplemented with 0.2 equivalents of 10 mM of ApNHp in 10 mM MgCl2. (f) Base specific contacts made between mCpol and the adenine base and corresponding electron density. (g) Base specific contacts made between Cas10 (Cmr2, PDB ID: 3W2W) and the adenine base and corresponding electron density.