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Figure 2
Procedure for on-chip crystallization on Roadrunner I chips. (a) For a crystallization experiment, the top of the chamber is sealed with transparent tape before the chamber wells are filled with ≥500 µl reservoir solution through the side opening. (b) The protein solution is mixed with precipitant solution and about 2–3 µl of this crystallization mixture is applied to the chip surface. (c) The chips are inserted into the crystallization chamber from the side and are held in place by a magnetic ring attached to the lateral opening of the chamber that interacts with the magnetic base of the chip. (d) Crystal-growth kinetics are monitored by light microscopy using top light illumination. (e) When crystal growth is complete, the chips are taken out of the chamber using a magnetic wand. The excess growth solution is removed through the membrane pores of the chip using a soft tissue, as described by Roedig et al. (2015BB39, 2016BB37). During all handling steps, starting from the removal of the mother liquor and until room-temperature measurements at the beamline are finished, it is essential to keep the crystals hydrated in a stream of humidified air (Sanchez-Weatherby et al., 2009BB42; Roedig et al., 2016BB37). The optimal level of applied humidity has to be tightly controlled and investigated beforehand for each protein individually, as this depends on the buffer composition (Wheeler et al., 2012BB51). For protein buffers that only contain low concentrations of salt and/or PEG, 98–100% humidity is usually suitable. (f) As an alternative to room-temperature measurements, crystals on a chip can be flash-cooled by plunging the chip into liquid nitrogen prior to cryogenic data collection (Roedig et al., 2015BB39).

Volume 6| Part 4| July 2019| Pages 714-728
ISSN: 2052-2525