Automated harvesting and processing of protein crystals through laser photoablation.

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

A simple and effective calibration method to determine the accuracy of liquid-handling nano-dispenser devices.

The accurate delivery of small volumes is a critical factor in the crystallization of macromolecules as it influences the reproducibility of the screening experiments. Crystallographic screening technologies have made it possible to perform experiments using volumes as low as 50 nl. The accuracy of the dispenser has usually been calibrated by weight measurements. In this work, a simple and inexpensive fluorescence-based calibration method that is sensitive and that can be used to monitor the precision and accuracy of any liquid-handling nano-dispenser device is presented. The results suggest that the protocol described here can be useful to determine volumes ranging from 50 to 300 nl with precision. Therefore, the pipetting of volumes as low as 50 nl can be calibrated periodically to ensure that precision and accuracy are maintained. The suggested calibration protocol can be executed in 6 h per instrument, including the calibration curve, which is the most time-consuming step; the rest can be completed in approximately 2 h.