Growth of large protein crystals by a large-scale hanging-drop method

A method for growing large protein crystals is described. In this method, a cut pipette tip is used to hang large-scale droplets (maximum volume 200 µl) consisting of protein and precipitating agents. A crystal grows at the vapor-liquid interface; thereafter the grown crystal can be retrieved by droplet-droplet contact both for repeated macroseeding and for mounting crystals in a capillary. Crystallization experiments with peroxiredoxin of Aeropyrum pernix K1 (thioredoxin peroxidase, ApTPx) and hen egg white lysozyme demonstrated that this large-scale hanging-drop method could produce a large-volume crystal very effectively. A neutron diffraction experiment confirmed that an ApTPx crystal (6.2 mm³) obtained by this method diffracted to beyond 3.5 Å resolution.