Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

This protocol provides the opportunity for scientists to characterize protein crystals embedded in highly viscous materials, as well as other soft materials currently hard to characterize using existing setups. Using this method, the samples are prepared in glass syringes, mounted directly onto the injector, making the injector system easy to use and minimizing sample waste. To prepare crystals at a high viscous medium, first centrifuge the crystal solution at low speed to form a soft crystal pellet.

After removing the excess medium, fasten a coupler to the end of one 100 microliter glass syringe and load 28 microliters of the crystal solution into the syringe barrel. Slowly insert the plunger into the top of the syringe and gently push the solution to the end of the coupler tip. To remove bubbles, use one gloved finger to apply very gentle pressure to the top of the blunted coupler needle point to create a seal and retract the plunger to draw the solution away from the needle tip to create a buildup of pressure within the syringe, then remove the finger to quickly release the pressure and to burst any bubbles within the syringe.

Alternatively, hold the syringe in one hand with the needle facing upwards and the plunger wedged between two fingers and quickly rotate the arm holding the syringe in one direction two to three times. The resulting centrifugal force will force any air bubbles out of the syringe. When all of the bubbles have been removed, use a fine spatula to add 42 microliters of high-vacuum silicone grease directly to the top of a second 100 microliter syringe and depress the plunger all the way to the end to remove any air bubbles and to ensure that there is no air gap in the end of the syringe.

When all of the bubbles have been removed from both syringes, holding both syringes by the ends, attach the syringes with the coupler. To mix the samples together, gently sequentially depress both the crystal solution and silicone grease syringe plungers 50 to 100 times until the materials are mixed thoroughly and appear to be homogenous. Then visualize the crystals under an optical microscope.

To determine the crystal concentration in the silicone grease, count the number of crystals in a specific area using the optical microscope images. To decrease the crystal concentration, repeat the crystal preparation using a higher volume of silicone grease or dilute the crystal pellet in crystallization buffer prior to the syringe setup. To increase the crystal concentration, repeat the crystal preparation using a smaller volume of silicone grease, being mindful not to reduce the viscosity below 10 pascal seconds.

When the appropriate crystal concentration has been obtained, move the entire sample into a single syringe and replace the coupler with a 108 micron inner diameter injection needle screwed very firmly into the base of the syringe. To set up the injector, select tools and homing mode and negative limit switch and start homing and let the software run until the screw is retracted sufficiently for a 100 microliter syringe to fit under the screw. Insert the needle of an empty syringe through the slit in the syringe holder to mount the syringe onto the injector and line the syringe against the bracket.

Wrap one O-ring across the mid section of the syringe, attaching the ring to the hooks on either side of the syringe and loop a second O-ring around the hooks on the upper section of the syringe. Make sure one part of the second O-ring is positioned on top of the glass syringe. To prepare the sample syringe, prime the syringe, place the injector cap on the sample syringe plunger head and load the syringe onto the injector.

Move the drive screw to the top of the cap. In the injector control software, select velocity mode and activate velocity mode. Set the setting value to 3, 000 revolutions per minute and click apply setting value.

As the screw approaches the cap on the syringe plunger head, change the setting value to zero. Once contact is made, click apply setting value to activate the preset value, stopping the screw instantly. Then gently wiggle the cap to make sure that it is firmly held in place.

To run the injector, change the setting value to 100 revolutions per minute and visually inspect the tip to observe the sample as it begins to extrude. After closing the hutch, decrease the setting value by decrements of 10 until a slow but stable stream of material is observed. If the sample stream does not flow well, increase the setting value in 10 revolutions per minute increments until the stream gets straighter.

If the stream does not stabilize, insert a polystyrene sample catcher under the sample stream to help guide the sample. Alternatively, insert a Venturi suction funnel into the sample catcher, which can be connected to the air outlet tube located in the hutch of the Australian synchrotron. To determine the flow rate of the stream, use the Lipidico device flow calculator.

Determining the optimal sample running speed for the injector is crucial for the stream stability. A slow flow rate results in an expansion of the silicone grease extruding from the injector, while a faster flow rate produces a thinner stream. Curling of the viscous media stream has regularly been observed.

To overcome this issue in highly charged samples, a polystyrene catcher can be used to introduce a weak electrostatic force. In this analysis, a glass syringe containing 26 microliters of lysozyme crystals suspended in silicone grease was used. A sufficient amount of data was collected within 38 minutes enabling the generation of the electron density map surrounding the disulfide bonds in the lysozyme structure.

Remember to use a small volume of sample on the glass slide to assess the crystal concentration of the sample to determine whether the viscosity needs to be corrected. The versatility of the injector allows the x-ray characterization of other highly viscous materials. For example, ionic liquid structures using battery research or colloidal materials using dairy products.