Scheduling the crystallization experiments:
The CORE user account allows the researcher to schedule the crystallization experiments within the crystallization calendar and thereby reserve date/time and specify the number of plates and setup temperature. This will ensure PCC is ready process your protein without any delay. When booking the crystallization experiments please keep in mind that PCC's capacity is limited to a maximum of 16 plates per half day.
Setting up the crystallization experiments:
The CORE user account allows the researcher to access the RockMaker application via the Citrix Workspace. Therein the crystallization experiments can and must be setup prior to delivering the protein samples.
For new researchers, a personal login for accessing the RockMaker application is created during the first visit to PCC. At the same time, the researcher receives instructions on how to efficiently and effectively set up and organize the crystallization experiments using the RockMaker application.
Within the Rockmaker application the following information can be specified:
Screen and Reservoir solution:
All initial crystallization screens offered by PCC are stored in deep well master blocks at 4°C and are available to the researcher for their experiments. Custom screens can be prepared delivered by the researcher or via the service offered at PCC. The latter requires the researcher to deliver the stock solutions in, for example, 50 ml Falcon tubes 3 days prior to the scheduled crystallization experiments. “Read-to-go” crystallization screens provided by the researcher have to be in a 96-well standard format and can be delivered the same day with the protein.
A Tecan EVO liquid handler is used to transfer 75 ul of each the 96 screening solutions from the deep well master block into the reservoir wells of the crystallization plate.
The standard plates used for all vapor diffusion crystallization experiments are either the SWISSCI UVXPO 2 Lens crystallization plate (Cat# UVXPO-2LENS) or the ARI Intelli-Plate 96-3 LVR (Cat# 102-0001-03) (Material/Equipment).
Setting up the nano-drop experiments:
The standard setup temperature of the crystallization experiment is room temperature (~21°C). For temperature labile protein samples the researcher can request a setup temperature of 4°C. It is essential that the researcher already specify the setup temperature in the crystallization calendar.
Depending on setup temperature, one of two nano-drop liquid handlers – Phoenix (20°C) or Gryphon-LCP (4°C) – is used for pipetting the vapor diffusion crystallization experiment.
The researcher also specifies the drop volumes for each of the two or three crystallization wells. This volume generally ranges between 100nl and 250nl for the protein sample and the reservoir solution. The researcher can, for example, explore the protein/mother liquor ratio as a screening variable.
Depending on the selected crystallization plate up to two or three different protein samples can be dispensed in one crystallization plate.
The protein sample volume required for setting up crystallization plates can be found in Sample Volume.
A Crystal Clear sealing tape from Hampton Research (Cat# HR4-506) seals all sitting drop crystallization experiments prior to incubation at 4 °C or 20 °C as specified by the researcher. A confirmation email will inform the researcher upon successful incubation.
The following review provides a summary of the current methods and technologies applied in automated platforms for the setup of initial and follow-up crystallization experiments:
P. Shaw Stewart and J. Mueller-Dieckmann
Automation in biological crystallization.
Acta Cryst. (2014). F70