CubeCrystal Plates for CIMP Crystallization

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

 

CubeCrystal Plate precoated with mono-olein (pink)

    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.

 

 

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.

 

 

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).

  

 

Bacteriorhodopsin (BR) crystal on CubeCrystal Plate

    
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.
 

 

Phase diagram in CubeCrystal Plates

    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.
 

CubeCrystal Plates: successful crystallization in LCP and lamellar phases

Fig. 4: In meso phases obtained dependent on water content.


 

 

Get started with CIMP in seven easy steps

 

  CubeCrystal Workflow Step 1: The Product    

Step 1: The CubeCrystal plate is based on the MRC 2-well UV transparent plate, with tiny lipid drops already predispensed into the protein wells. 

  
  CubeCrystal Workflow Step 2: Dispense Protein  

Step 2: Dispense 100 nl of the protein solution using a standard nanoliter robot.

Use protein concentrations of 2-5 mg/ml as starting point.

  CubeCrystal Workflow Step 3: Seal and incubate  

Step 3: Seal the plate using your favorite sealing tape. Incubate for 1-3 hours to allow the meso phase to form. 
 

  CubeCrystal Workflow Step 4: Dispense Precipitant  

Step 4: Dispense the precipitant into the reservoir with a manual pipette or microliter liquid handler.

Avoid solutions containing alcohols, organic solvents, and high concentrations of PEG.

  CubeCrystal Workflow Step 5: Dispense Precipitant 2  

Step 5: Dispense 100 nl of the precipitant solution into the protein well with a nanoliter robot. Try different dilutions of the precipitant solution to reach different meso phases in the vapor diffusion experiment (Fig. 3)
 

  CubeCrystal Workflow Step 6: Seal and incubate   Step 6: Seal the plate again, and incubate it at a suitable temperature, typically 18-22°C for mono-olein plates.
  CubeCrystal Workflow Step 7: View and harvest  

Step 7: Look at your plate in regular increments to  monitor crystal formation.

Crystal harvesting can be facilitated by using X as cryoprotectant.



 
Literature reference
 

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

  • (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

    Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

    Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

  •  
 

 

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