Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

The overall goal of this procedure is to prepare samples of membrane protein crystals in lipidic cubic phase for serial crystallography data collection at synchrotron and x-ray-free electron-laser sources. This method should help answer key questions in the field of structural biology of membrane proteins, such as transport and single transduction across the membrane. The main advantage of this technique is that it enables structure determination of membrane proteins in their native lipid environment at room temperature with minimal sample consumptions and a negligible radiation damage.

Individuals new to this method will struggle because lipidic cubic phase is a viscous gel-like material that is difficult to handle without special tools and prior experience. Begin this procedure with the reconstitution of membrane proteins in the Lipidic Cubic Phase, or LCP, using syringes number one and number two, as described in the text protocol. After a transparent and homogeneous LCP is formed, move the entire sample into Syringe Number Two.

Detach Syringe Number One while keeping the coupler connected to Syringe Number Two. Connect a removable needle to another 100-microliter clean syringe, called Syringe Number Three. Aspirate approximately 70 microliters of the precipitant solution into it.

Disconnect the needle from Syringe Number Three while keeping the Teflon spherule inside the syringe. Connect Syringes Number Two and Number Three through the coupler, making sure that Teflon spherules are correctly in place in both syringes. Carefully screw the coupler tightly into position.

Orient the coupled syringes vertically, with Syringe Number Two on the bottom, and inject the protein-laden LCP sample from Syringe Number Two into Syringe Number Three slowly and steadily, until the LCP string touches the plunger of Syringe Number Three. The injected LCP volume will amount to about 1/10th of the initial precipitant volume in Syringe Number Three. Verify the volume by the scale reading on both syringes.

Then, disconnect Syringe Number Two. The coupler now is only connected to Syringe Number Three, which contains the sample immersed in the precipitant solution. Use Parafilm to completely seal Syringe Number Three, including the plunger-syringe interface, the opening end of the coupler, and the needle nut.

Incomplete sealing during this step can cause the precipitant conditions to change and the sample to dehydrate. Repeat the injection of the protein-laden LCP sample steps to set up crystallization in six additional syringes, utilizing a total of approximately 50 microliters of the LCP sample. Store the sealed syringes in a plastic sealable bag with one or two fiber-free cleaning tissues presoaked with water to protect against sample dehydration.

Seal the bag, and store the syringes in a 20-degree-Celsius incubator during crystal growth. For crystal detection, remove the syringes from the incubator to emit the LCP samples directly inside the syringes every 12 to 24 hours under a stereo microscope with cross-polarized light. Identify crystals as tightly-packed shiny particles, or, in the case of smaller crystal size, as a uniform glow from the LCP filament.

Return the sealed syringes to the bag, and store at 20 degrees Celsius for future use. The lipid titration step is necessary to absorb the excess precipitant solution and to prevent lipid freezing upon injection into the x-ray-free electron laser beam. About one hour before the beginning of data collection, remove the syringes with the samples from the 20-degree-Celsius incubator.

Select two to four syringes for sample consolidation based on a similar crystal appearance and similarity of crystallization conditions. Carefully remove the Parafilm sealing from the selected syringes. Remove the syringe coupler, and attach clean removable needle to the first selected syringe.

Gently and slowly push the plunger forward to squeeze the precipitant out through the needle into a microcentrifuge tube. Exercise caution at this step;because applying high pressure on the plunger can eject some of the crystal-laden LCP along with the precipitant solution, leading to a partial or complete loss of the sample. Stop the plunger when most of the precipitant has been removed and the LCP has accumulated at the needle entrance.

Perform this process for the other selected syringes. To consolidate the resultant LCP samples, connect two syringes together through a clean coupler. Depress the plunger on one syringe to transfer the entire sample from one of the syringes to another.

Disconnect the empty syringe. Repeat this step to consolidate all of the crystal-laden LCP material into one syringe. Remove as much precipitant as possible.

Add approximately five microliters of 7.9 MAG or the original shorter-chain MAG host lipid to an empty syringe. Connect this syringe to the syringe with the consolidated sample through a syringe coupler, and mix by alternatively depressing the syringe plungers. Repeat the process until all the residual solution has absorbed and a homogeneous and transparent LCP is formed.

Move the entire mixed LCP sample into one syringe, and disconnect the empty syringe. Attach an LCP injector loading needle to the syringe with the consolidated sample. Carefully eject approximately one microliter of the LCP sample on a glass slide, and cover it with a glass cover slip.

Gently press on the cover slip to sandwich the sample. Take images of the LCP sample under a stereo microscope at the highest possible magnification, using a bright field illumination and cross-polarizers. If possible, take additional images using a UV florescence microscope and imager to confirm the existence of protein microcrystals in the sample.

Estimate crystal size and density. The ideal crystal density for a data-collection experiment will depend on the crystal size, the diameter of the x-ray beam, and the diameter of the LCP stream, and should result in a crystal hit rate of about 10 to 40%Finally, load the LCP injector, and perform LCP serial femtosecond crystallography, or LCP SFX data collection, as described in the text protocol. Shown here are microcrystals of the serotonin receptor in complex with ergotamine imaged using a bright-field microscope mode, and imaged using a cross-polarized light microscope mode.

The structure of the serotonin receptor ergotamine complex was obtained by the LCP SFX approach. Shown here are microcrystals of the smoothant receptor in complex with cyclopamine that were imaged using a cross-polarized microscope mode. The structure of the receptor cyclopamine complex was obtained by the LCP SFX approach.

Once mastered, this procedure can be done in less than two hours if it is performed properly. Though this method can provide insight into the structure of membrane protein targets, with slight modification, it can also be applied proteins. Implication of this technique extends towards drug discovery;because high-resolution structures provide excellent templates from the standard lignan protein interactions and help with designing more efficient drugs.