Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

This protocol has been designed to ensure that problems frequently encountered during time-resolved serial crystallographic experiments won’t compromise data quality. One major advantage of this method is its adaptability to individual crystal systems. The high speed camera extrusion test also allows us to directly measure extrusion stability.

First, load 50 microliters of crystalladen monoolein based LCP in a 100 microliter syringe about 30 minutes before the injection. For injection into a vacuum environment, load five microliters of MAG 7.9 and five microliters of liquid paraffin into the back of a second syringe. Holding the syringe vertically, expel the air bubbles from the syringe.

Connect the syringe with MAG 7.9 and paraffin to a standard syringe coupler. And purge the air from the coupler by gently pressing on the plunger until a small volume of the mixture is visible at the tip of the coupler needle. Connect the sample syringe to the syringe coupler taking car not to introduce any air into the sample.

Mix in the lipid and paraffin by passing the sample through the coupler multiple times. Next, load 20 microliters of premixed LCP in another 100 microliter syringe. Remove the empty syringe from the coupler and attach the premixed LCP to the crystal containing syringe using a standard syringe coupler.

Then, pass the sample through the coupler 100 times. To bring the sample into the cubic phase, add three microliters of monoolein and mix 50 times. Repeat this procedure just until a transparent phase has formed to avoid an excess of monoolein.

As a preliminary test for sample stiffness and extrudability, detach the empty syringe from the syringe coupler, and holding the syringe vertically, squeeze a small amount of sampler through the coupler. If the extruded sample forms an upright cylinder, then the sample is ready for extrusion testing. Adjust the total volume of the sample to 100 microliters by adding more premixed LCP.

Attach the sample syringe and two empty syringes to the three-way syringe coupler. Mix at least 50 times by passing half of the sample into the second syringe and then pressing both halves of the sample into the third syringe simultaneously. Place the syringe containing the mixed sample under a stereo microscope to verify a homogenous distribution of crystals.

Purge an HPLC pump and all water lines to ensure the flow rates are accurate. Then, purge the hydraulic stage of the injector. Next, turn on and connect the camera to the supplied software.

With a live video running for visual feedback, position the nozzle tip in the center of the frame and bring it into focus with the three-axis stage. Set frame rate on the camera to 1, 000 frames per second. Then, set the resolution to 512 by 512 pixels.

With the exposure time now set by the frame rate, adjust the illumination level until the nozzle is visible. Reposition the nozzle tip so that it centered to left to right and is located in the top third of the frame. Set up the camera in time lapse mode.

Set the interval to 30 seconds and the repeats to 40 times. Set the trigger mode to random. And enter the number of frames to record to 1, 000.

Now, load the reservoir with 20 microliters of the test sample and attach the capillary nozzle. Attach the filled reservoir to the injector. Then, attach the gas line to the port on the nozzle and start the gas flow.

Following this, simultaneously start the pump and the camera recording. Monitor the extrusion until the pump pressure sharply ramps up near the expected end time. Then, stop recording, shut off the pump, and vent the system pressure by opening the relief valve.

Following this, open the video file with the analysis software and calibrate the measurement tools. Find a frame in the video where there is a trackable feature visible in the extrusion. Record the frame number.

Next, advance the video to a frame where the same feature is visible but has moved from its position in the previous step. Record the frame number. Using the straight line measurement tool, measure the distance from the feature’s start and end point via analyze measure.

Repeat the previous steps a few times for every segment of the video. Finally, plot the data series. The ideal starting material for the procedure described here is high densities of microcrystals incorporated into a viscous carrier medium for the injector.

Protein crystals were used to collect TRSFX data on the proton pumped bacteria rhodopsin, which revealed the ultra fast changes that occur after photon absorption. After sample preparation using a three-way coupler, visual inspection of the material in the syringe shows sample homogeneity. And microscope images can confirm the density of the crystals.

The sample is in the cubic phase when the delivery medium is clear and viscous. Turbid mixtures are an indication that the sample is in a sponge or lamellar phase, but are not conclusive as high crystal density may obscure the LCP’s clarity. A low pressure test to identify the sponge phase can be performed by pulling the syringe plunger away from the sample.

During the jet test, the sample should extrude a long continuous column of LCP that moves at a nearly constant velocity. Samples that run in a dripping mode indicate that the viscosity is too low. Data from samples that form a column should show that the extrusion stays above a minimum speed dictated by the experimental parameters.

After optimization using this protocol time-resolve serial crystallographic data can be collected at an expert facility. This will reveal the conformational changes in entire protein as it performs its function.