The crystallization laboratory component of the Macromolecular Crystallography Laboratory is located in room 2750 of the Stephenson Life Sciences Research Center on the University of Oklahoma-Norman South Research campus. The mission of the laboratory is to help researchers at the university and throughout the State of Oklahoma crystallize protein and/or nucleic acid samples of interest.
Funding for the robotic instrumentation was provided by the National Science Foundation, Award 0922269.
The Macromolecular Crystallography Lab (MCL) provides nano liter volume crystallization screening using the Mosquito by TTP Labtech. The Mosquito uses a continuous reel of disposable positive displacement micropipette tips capable of dispensing volumes ranging from 50 nL to 1.2 µL. The use of a disposable pipette guarantees no cross contamination. Currently, the facility offers a number of commercial 96 and 48 condition screens. Please ask the Facility Manager what screens are available. You may supply a non-commercial screen or a commercial screen that we do not have. If using a screen not offered by the MCL, you will be responsible for filling the tray, or at the very least a 96 well block, to be used on the Mosquito. Please contact the Facility Manager if you have questions regarding the use of your own screens.
The minimal volume of sample needed to set up a 96 well tray is 15 µL. This will allow pipetting of 100 x (50 nL-1200 nL) drops over 96 conditions. Optimum drop sizes and initial screens can be determined in consultation with laboratory personnel. It is recommended that if you have the purified compound to use that 300 nL x 300 nL be set up. Successful trials can then be optimized in the crystallization laboratory or at the researchers home laboratory.
All crystal trays will be imaged at room temperature automatically by our Rigaku Americas Desktop Minstrel with Gallery 160 imaging system. Along with the imaging system the Alchemist II fluid handler also from Rigaku is available for the production of optimization/fine screens. The DT Minstrel and Alchemist II are integrated through the Crystal Trak program suite.
The MCL can also provide guidance and materials to users in setting up crystallization trials by hand. It is recommended that only 24 or 48 well trays be done in this manner and all 96 well trays be set up by the Mosquito.
Upon the successful crystallization of the macromolecule of interest the laboratory will then provide guidance in screening of the crystals for diffraction and subsequent structure solution.
Applicable fees will be charged for services.
Consumables that can be provided include:
- 96 and 24 well crystallization trays
- crystallization reagents
- materials for sealing tray wells
- additives for screening and optimization
Equipment provided for conducting crystallization trials:
- pipetters and other needed glassware
- incubators for temperature control
- microscopes for visualization and recording of trial wells
Samples submitted for crystallization trials should be as homogeneous and pure as possible in order to have the highest probability for success. All biochemical and biophysical information that can be obtained can aid in the prediction of successful crystallization. At the very minimum an overloaded SDS-PAGE gel should show a single band at the correct molecular weight for the compound. Other tests that can aid in determining purity and homogeneity:
- Size Exclusion Chromatography (SEC)
- Mass Spectroscopy
- Isoelectric Focusing (IEF) gels
- Amino Acid Analysis
- N-terminal sequencing
- Ultra centrifugation
- Dynamic Light Scattering
The other major physical property to consider for the sample is concentration. A general rule of thumb is to have a sample concentration of 10 to 20 mg/mL. If the sample can be concentrated to much higher levels then do so and, conversely, if the sample cannot be concentrated to that level then it should be concentrated to a level just under where the sample will aggregate or precipitate out.