The 3Dpol proteins all contained the solubility-enhancing
L446D/R455D mutations on the thumb domain [10] and
were expressed, purified, and crystallized as previously
described [13]. Crystals were transferred to 4 °C in
Hampton micro-bridges and slowly equilibrated into a
final solution containing 250 mM sodium acetate, 30% (w/
v) polyethylene glycol 400, 0.1 M cacodylic acid (pH 7.0),
and 2 mM DTT. NTP soaks were carried out using this
same solution supplemented with 10 mM NTP and 10 mM
MgCl2 for ~ 1 h, and crystals were flash frozen with liquid
nitrogen.