We support our clients in developing SBDD programs by offering a stand-alone protein crystallization service. It does not require a large quantity of protein (in most cases 5 mg is sufficient) and provides a quick and cost-effective assessment of protein or protein complex crystallizability.
The service includes the preparation of the samples for crystallization experiments, setting up 8 commercial sparse matrix screens at two temperatures, and monitoring the plates for crystal growth. The experiments are set up using a Gryphon nanodispensing robot in a 96-well format and runs are scheduled in advance on a monthly basis. The protein target and compounds of interest should be provided by the client.
Please, find the information on the sample requirements below together with more detailed information on the experiment design.
Sample requirements for crystallization experiments.
- 500 μL of the protein aliquoted in 100 μL/tube and frozen in 0.2 mL thin-wall PCR tubes;
- High quality and purity (≥ 90%), non-aggregated, high-quality, homogenous, and stable samples;
- Concentration of 10 mg/mL is a good starting point for the majority of soluble proteins; however, lower concentrations can be used if solubility and protein concentrating are problematic;
- All additives important for stability should be added to the storage buffer. We recommend decreasing glycerol concentration to 10% (w/v) or lower;
- Avoid using detergents such as Triton X-100 or Tween20, and feel free to contact us to verify the storage buffer composition for crystallization screening;
- Lower the storage buffer concentration to 25 mM to minimize its contribution during crystallization;
- Snap-freeze your samples in liquid nitrogen and store them at -80 °C;
- If possible, please, provide us the following information about the protein:
|Identity/UniProt number – if possible|
|Construct design with construct boundaries|
|Protein concentration (in mg/mL or in μM)|
|Storage buffer content|
|Comments on storage and stability|
- The compound of interest should be optimally soluble at 10 mM in 100% DMSO and at 1 mM in the protein storage buffer with 10% (v/v) DMSO. Lower solubility, depending on the compound-protein affinity may hinder the generation of protein-ligand crystals;
- The compound might be provided as:
|Pre-formed complex with the protein of interest;|
|DMSO stock (ideally 10 mM in 100% (v/v) DMSO or as high as possible);|
|Powder (2-3 mg)|
Important: in order to avoid possible damage to the equipment and comply with safety rules, we ask you to provide MSDS for compounds and/or possible safety issues working with samples and compounds.
Shipping the sample.
We recommend shipping the frozen samples in dry ice by DHL or CryoPDP. If the samples are sensitive to freezing, please contact us to find an alternative solution.
- Upon arrival, the protein will be immediately transferred and stored at -80 °C;
- On the day of crystallization screening, the protein will be quickly thawed and centrifuged to remove any residual precipitation;
- The compound of interest will be added to the protein solution at 1 mM final concentration, but not higher than 10% (v/v) DMSO concentration, and incubated for 30 minutes on ice;
- In case protein or compound precipitates, the samples will be centrifuged and the final protein concentration will be measured;
- Right before crystallization, all samples will be filtered through Ultrafree-MC (0.5 mL, 0.1 μm) Centrifugal Filter Units;
- The crystallization screening will be set up using the sitting drop vapor diffusion technique in 96-well 3-drop conical crystallization plates (Swissci) with Crystal Gryphon Nanodispenser (Art Robbins Instruments) and commercially available crystallization screens (Salt-Rx (HR2-107, HR2-109), Index (HR2-144), PEG-Rx (HR2-082, HR2-084), Morpheus (MD1-46), JCSG-plus (MD1-37), PACT premier (MD1-29), BCS (MD1-104) and Ligand Friendly (MD1-121);
- 200 nL of prepared complexes will be mixed with 200 nL of the reservoir solution and equilibrated against 40 µL of the reservoir solution at 4 or 18 °C;
- The crystallization plates will be examined for the presence of crystals after 2 and 4 weeks;
- (Optional and subject to a separate quotation) Crystals suitable for X-ray measurements will be treated with various cryoprotectant solutions, flash-frozen in liquid nitrogen, and evaluated by X-ray diffraction.