Protein Crystallization Core Facility
The Protein Crystallization Core Facility sets up and monitors crystallization trials at nano-litre scale to help structural biologists and other interested scientists to progress their work on structural characterization of the proteins and biological complexes of their interest.
The Protein Crystallization Core Facility provides state-of-the-art structural biology infrastructure for researchers working at the Malopolska Centre of Biotechnology as well as co-operation partners or researchers outside MCB (both form academia and the industry).
To facilitate obtaining crystals suitable for X-ray diffraction studies,
we would offer a wide range of crystallization services:
Initial screening
searching for the best crystallization conditions yielding protein mono-crystals. We routinely test over 1700 various conditions.
Refinement screening
improving the initial conditions by use of additives (kosmotropic, chaotropic, salts and other additives)
Anaerobic crystallization
increasing the probability of crystal growth by using anaerobic chamber
Custom services
consulting expertise and carry out of various crystallization experiments
Service cost
Standard crystallization condition screening is performed in 96 well plates using 200-1000nl drops (different setups are available on request, ex. MRC plates, Linbro Plates, hanging drop). The service is charged “per plate” (80PLN brutto for internal MCB users / direct collaborations; please contact us for the prices for external users).
Example: The user wishes to screen her/his protein using ~1000 initial conditions. 10 plates will be set (960 conditions) with the provided protein at the cost of 10*80PLN=800PLN
Initial screening
We offer about 20 crystallization screens (see AVAILABLE SCREENS) of commercial origin which allow you to test a wide variety of initial screening conditions. All of the screens are formatted into 96 wells (8 x 12 array). The crystallization experiments are based on vapour diffusion method and are designed for up to 192 nano-scale experiments for one plate. Our robot (Phoenix) transfers 50 µl of the 96 screening solutions from a deep well master block into the reservoir wells of the crystallization plate. The researcher specifies the drop volumes ranging between 200-1000 nl for the protein sample and for the screen solution. Up to two different protein samples or concentrations can be dispensed in a single well of the crystallization plate (two well pedestals). Alternatively, you can use the two wells available at each sample pedestal to explore the protein/mother liquor ratio as an additional screening variable. Varying the drop ratio will change the initial and final sample concentration and the initial reagent concentration and also alter the equilibration kinetics between the drop and the reservoir. The standard setup temperature of the crystallization experiment is room temperature. In our crystallization room, where we set up and keep crystallization plates, we maintain constant temperature 22°C. For temperature labile protein samples (or other reasons – ex. exploring an additional variable) the researcher can request 4°C as the setup temperature of the crystallization experiment.
Required Sample Volume
The minimal sample volume per one drop (using the Mosquito robot) is 25 nl, i.e. 2,5 ul per plate. For technical reasons, total volume of the sample needs to exceed by at least 2µl the calculated volume for all plates.
Refinement screening
In order to fine-tune successful hit conditions and to grow larger and/or better-diffracting crystals, take the initial conditions identified by screening are optimized by varying the concentration of the precipitant as well as the buffer pH and/or other parameters in a systematic manner.
We can save your time and chemicals by provide custom crystallization screens with our design or your design and our preparation. We prepare arrays of up to 96 conditions in 0.5-1.5 mL aliquotes.
In our platform, a wide range of in-house stock solutions is available. To make sure that we have all the stocks for your refinement screen please contact us before ordering.
As a part of the refinement screening we also offer an additive screening. Additive screen is used for the optimization of preliminary crystallization conditions where special additives may assist in the crystallization of the sample and improve the quality of the crystal. The 96-additives kit which we use, focuses mainly on small molecules which are known to modify the sample-sample and sample-solvent interactions, and/or modify the surface water envelope.
Users interested in the optimization of obtained crystals may also benefit from the seeding procedure.
We offer help in two kinds of seeding experiments: classical microseeding and heterogenous seeding.
Microseeding
When your crystals grow with unsatisfactory morphology, have low resolution or poor nucleation rate we offer microseeding. The poor quality crystals are crushed and used to seed the nucleation points in a new crystallization experiment. The standard protocol setups use drops: 0.3µl protein, 0.2µl reservoir solution, 0.1µl seed stock. You can change drop ratio to get the better crystals from the seed stock.
Heterogeneous seeding
If poor nucleation rate is encountered (ex. no crystals in the initial screening effort) heterogeneous seeding becomes an opportunity. Seeds of heterogeneous material (ie. ceramic seeds) are introduced into the crystallization drops to offer additional nucleation sites for your crystals.
Anaerobic crystallization
Our services include the possibility of performing crystallization in anaerobic conditions. Our custom-made gloveless anaerobic chamber offers the operator a convenient glove-free ability to handle and inspect samples under low oxygen (0-5 ppm) conditions. Moreover, as it encapsulates our Mosquito crystallization robot, it gives the user the capability to take advantage of nano-litter dispensing in a low oxygen environment.
Custom services
Our Core Facility offers consulting expertise and carry out of various crystallization experiments including: pre-crystallization tests, 24-well crystallization plates in hanging drop setup, 48 well crystallization plates, seed beads. We are also open for any non-standard inquires and requests. Please work out with us the solutions suited for your particular needs.
All interested are welcome to contact us at: service@structuralbiology.pl
Material and equipment
• Phoenix Liquid Handling System (Art Robbins Instruments)
To set up your plates we use a multiposition, high-throughput liquid-handling platform. This unique instrument combines the precise low-volume dispensing capabilities of a syringe head with an accuracy and speed of the on-the-fly non-contact nanodispenser. Both the 96 syringe head and the nanodispenser have a large volume range. You can specify a drop size and a ratio of protein to screen within each drop.
• Crystallization plates
The standard plates used by us for all vapour-diffusion crystallization experiments are the SWISSCI MRC 2-well crystallization plates. They allow to set up to 192 nano-scale experiments per single plate (96 conditions, two sample pedestals per condition). Other plates are available on request.
• Microscope – Leicia M125
You can control and observe crystals growth by our microscope Leicia.
• Dewars for crystal transport and storage
In our liquid nitrogen dewars is possible to storage and transport large quantities of crystals at cryogenic temperatures.