The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.
Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.
The protocols provide a basic visual guidance for the main steps involved in performing in meso crystallization experiments. More in-depth details related to these protocols, emphasizing possible pitfalls, shortcomings or alternative routes are available elsewhere1,2. Optional LCP-FRAP assays can help at the earlier stages to select the most promising protein construct, LCP host lipid and lipid additives, as well as limit the range of possible precipitants and buffer conditions3. Once an initial crystallization hit is found, it should be optimized to obtain better quality crystals. Optimization of in meso crystallization conditions is essentially similar to optimizing conditions for soluble proteins with the addition of extra parameters associated with the composition of LCP1. Membrane protein crystals grown in lipidic mesophase are typically smaller in size than crystals obtained in detergent solution, but more ordered, thus, benefitting strongly from using microfocus beamlines available at modern synchrotron sources6.
Many of the procedures related to in meso crystallization, including setting up crystallization or assay plates, conducting LCP-FRAP assays and detecting crystals, have been semi- or fully automated1,2,8,9, allowing screening of a large range of conditions while consuming small amounts of protein and lipid. On the other hand, protein reconstitutions in LCP and crystal harvesting remain manual and more tedious operations and, thus, have a need for improvement.
The authors have nothing to disclose.
This work was funded in parts by the NIH grants GM073197 and RR025336.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
100 μL gas-tight syringe | Hamilton | 7656-01 | 2 syringes are required | |
10 μL gas-tight syringe | Hamilton | 7653-01 | ||
Syringe coupler | Hamilton | 7770-02 30902 | The coupler can be made using available parts as described in refs. 10 | |
Repetitive syringe dispenser | Hamilton | 83700 | The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11 | |
Short (0.375”) flat-tipped removable needle (point style 3, gauge 26) | Hamilton | 7804-03 | ||
96-well glass sandwich plate | Marienfeld | 08 900 03 | For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12. | |
Glass cover slip | Electron Microscopy Sciences | 63787-01 | ||
monoolein | Sigma | M7765 | ||
Crystallization screens | Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience | Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13. | ||
Capillary cutting stone | Hampton Research | HR4-334 | ||
Fine point tweezers | Ted Pella | 510 | ||
Angled sharp probe | Ted Pella | 13650 | ||
MicroMounts | MiTeGen | M1-Lxx-xx | Select MicroMount diameter to match the crystal size |