Lipidic cubic phase crystallization is the most popular method for membrane protein structure determination. Experiments are typically done in batch format. However, we know from the crystallization of soluble proteins that vapor diffusion can be a lot more powerful. The Controlled in meso phase (CIMP) method (1) combines the benefits of LCP and vapor diffusion to push membrane protein crystallization to new limits. Cube Biotech offers ready-to use plates for an easy start with this revolutionary method.
Features of the CIMP technology:
- Fully automatable method, compatible with any nanoliter robot
- Combines LCP with vapor diffusion to increase success rate
- Uses low protein concentrations (2-5 mg/ml)
- Can be easily combined with lipid variations
Fig. 1: Overview of a CubeCrystal Plate. Lipids are visualized with a purple dye.
What is a CubeCrystal Plate?
CubeCrystal Plates come ready to use. They are based on the MRC 2-well Crystallization Plate made of UV transparent, advanced polymer. The two protein wells are coated with lipid, typically mono-olein of high purity
. The purified protein can be dispensed directly into the protein wells as in standard protocols for vapor diffusion.
How to image a CubeCrystal Plate?
CubeCrystal Plates can be visualized just like any vapor diffusion plate. See an image of bacteriorhodopsin crystals visualized on a Rigaku XtalDetectR(TM) instrument. Crystals from 2 - 60 µm in size could be visualized. Because of the UV transparent plastic, a UV detection is also possible to distinguish protein from salt crystals.
Fig. 2: Visualization of BR crystals in a CubeCrystal Plate. Image taken on a Rigaku XtalDetectR(TM) instrument. Crystal sizes range from 2-60 µm.
CIMP: the clever combination
With CIMP CubeCrystal Plates, you are setting up experiments that combine the most popular methods in protein crystallography.
Lipidic cubic phase, the most successful method for membrane proteins, is achieved in a gentle, isothermal manner, and extended to other in meso phases such as sponge and lamellar phases. Vapor diffusion, the most successful method for all kinds of proteins, is extended to lipidic cubic phase Counter diffusion: Areas of higher and lower concentrations of protein and precipitant form during the passive diffusion within the lipid. In fact, each drop contains a series of different protein:precipitant ratios, which increases the chance for crystal formation.
Fig. 3: Phases in the LCP experiment. Idealized diagram of phases forming in the lipid drop as a result of passive diffusion of protein and precipitant into the lipid phase. Crystals form in the nucleation/metastable phases.
Simply add water to walk through the phases
Varying the water content is a very easy way to optimize the crystallization experiment. By diluting the reservoir solution which is added to the protein well, you increase the concentration gradient between reservoir and protein well. Over the course of the vapor diffusion experiment, this decreases the water content in the meso phase. With the starting point of the experiment being constant at about 87% water, you can determine the end point of your experiment by choosing different dilution factors.
In meso phases that can be reached with the CIMP technology
Hydrated LCP phase: a modified lipidic cubic phase with water contents between 90 and 45%. Lipidic cubic phase (LCP): the classic in meso phase with water contents between 45 and 26%. Batch experiments typically are set up at water contents between 30 and 35%. Lamellar phase: characterized by increased contact between proteins. Water content below 26%. Crystal formation can happen in any of these phases. For our model protein bacteriorhodopsin, we obtained crystals of different cell geometry, size, and diffraction in the different phases (see Fig. 4).
Fig. 4: In meso phases obtained depending on water content.
|Name of method
||Controlled in meso phase (CIMP) method
||Plates made of UV transparent, advanced polymer
||Protein wells are coated with dry mono-olein lipid
|Protein drop volume
||100 nl protein solution + 100 nl reservoir solution
||50 µl reservoir solution
|Amount of coating lipid
||29 µg Mono-olein (1-(cis-9-Octadecenoyl)-rac-glycerol)
- Liquid handling robot capable of pipetting 100 nl volumes
- Liquid handling robot capable of pipetting 50 µL volumes - alternatively Micropipettor
- Micropipettor (preferably 8-channel pipette) to dispense 200-800 µL volumes
- Micropipetting tips
- F Temperature-controlled incubator for crystallization plates
- Microscope for visualization of protein crystals
- Crystallization screening solutions (e.g. 96x1.5mL)
- 2 sealing tapes per plate
- 1 empty deepwell block, holding 96 x 1.5 mL or 96 x 2 mL volumes (e.g. Corning 3961)
Get started with CIMP in nine easy steps
The product: The CubeCrystal plate is based on the MRC 2-well UV transparent plate, with tiny lipid drops already predispensed into the protein wells.
Step 1: Equilibrate the CubeCrystal Plate to room temperature by incubation for at least 10 min. Note: Do not unpack the plate before reaction setup.
Step 2: Prepare protein solutions concentrated 2 mg/ml to 5 mg/ml for first trials. Tip:For optimization, test two different protein concentrations in the two parallel wells.
Step 3: Prepare suitable dilutions of the precipitant solutions according to Table 1 using a microliter dispenser or a micropipettor, into the 96 wells of the spare deepwell block. Mix by pipetting up and down.
Step 4: Unpack the CubeCrystal Plate, and dispense 100 nL of the protein solution onto the dry lipid in each protein well using a nanoliter robot. Use protein concentrations of 2-5 mg/ml as starting point. Note: The CubeCrystal 2-well MO plate is based on the Swissci MRC 2-well plate, so that similar teaching protocols can be used.
Step 5: Seal the plate using your favorite sealing tape. Incubate for 1-3 hours to allow the meso phase to form. Note: It is crucial to incubate at no less than 18°C in all steps.
Step 6: Dispense 50 µL each of the 96 undiluted precipitant solutions into the reservoir wells of the CubeCrystal Plate using a micropipettor or pipetting robot
Step 7: Dispense 100 nL each of the 96 diluted precipitant solutions from the spare deepwell block into the corresponding protein wells of the CubeCrystal Plate using a nanoliter robot. (Fig. 3)
Step 8: Seal the plate again, and incubate it at a suitable temperature, typically 18-22°C for mono-olein plates.
Step 9: Monitor the plates on a regular basis using a microscope. During the first two weeks, a daily inspection is recommended. Crystal harvesting can be facilitated by using X as cryoprotectant.
(1) Kubicek J, Schlesinger R, Baeken C, Büldt G, Schäfer F, Labahn J (2012) Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins. PLoS ONE 7(4): e35458. doi:10.1371/journal.pone.0035458