Name: lipidico injection protocol for serial crystallography measurements at the australian synchrotron
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Description: Watch this Scientific Journal Video about Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron at JoVE.com

This work was supported by the Australian Research Council Centre of Excellence in Advanced Molecular Imaging (CE140100011) (http://www.imagingcoe.org/). This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector.

1. Preparation of crystals in a high viscous media using glass syringes
<ol>
	<li>Centrifuge the crystal solution gently (~1,000 x g, ~10 min at 22 &#176;C) to form a soft crystal pellet and remove the excess buffer. This will result in a high concentration of crystals in the pellet which can be used for data collection. 
	NOTE: To prevent dilution of the viscous media increase the crystal concentration at this step. Optimize the ratio of viscous media to crystal volume for each sample to obtain a high co...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+millisecond+crystallography+of+membrane+and+soluble+protein+microcrystals+using+synchrotron+radiation.">Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.</a> International Union of Crystallography. 4, 439-454 (2017).</li><li>Berntsen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+millisecond+crystallography+instrument+at+the+Australian+Synchrotron+incorporating+the+"Lipidico"+injector.">The serial millisecond crystallography instrument at the Australian Synchrotron incorporating the "Lipidico" injector.</a> Review of Scientific Instruments. 90 (8), 085110 (2019).</li><li>Botha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room-temperature+serial+crystallography+at+synchrotron+X-ray+sources+using+slowly+flowing+free-standing+high-viscosity+microstreams.">Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.</a> Acta Crystallographica Section D-Structural Biology. 71, 387-397 (2015).</li><li>DePonte, D. 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An alternative HVI has been developed, ideal for carrying out SX experiments at synchrotron sources. It has two key advantages over existing HVIs. First, it is easy to install on the beamline allowing rapid switching between conventional crystallography and SX, just ~30 minutes is required for installation&#160;and alignment on MX2. Second, the sample syringes used to grow crystals can be directly used as the reservoirs for injection, limiting wastage during sample transfer. The protocol for changing samples has been des...

Serial crystallography (SX) is a technique that was developed initially in the context of X-ray Free Electron Lasers (XFELs)1,2,3,4. Though fixed target approaches can be used for SX5,6,7, typically, injector systems are employed to deliver crystals in a continuous stream to the X-ray beam. Because it combines data from a large number of crystals, SX avoids the need for any crystal alignment during the experiment, and enables data to be collected at room temperature8,9. With the aid of a suitable injector, the crystals are streamed one-by-one into the X-ray interaction area and the resulting diffraction data is collected on an area detector9,10. To date, SX has been successful in solving a number of protein structures1,11,12,13 including crystals too small to measure using conventional crystallography. It has also provided new insights into time-resolved molecular dynamics by exploiting the femtosecond pulse duration of the XFEL. By initiating pump-probe reactions with optical laser sources, in-depth studies have been carried out on photosystem II14,15, photoactive yellow protein16,17, cytochrome C oxidase18, as well as bacteriorhodopsin19,20,21. These studies have probed the electron transfer dynamics that occur following light activation demonstrating the significant potential of serial crystallography for understanding time-resolved biological processes.
Development of serial crystallography is also becoming increasingly prevalent at synchrotron sources9,12,20,22,23,24. Synchrotron based SX allows for large numbers of individual crystals to be measured efficiently at room temperature using an appropriate injector system. This approach is suitable for smaller crystals hence, in addition to requiring a fast frame-rate detector to collect the data, a micro-focused beam is also required. Compared to conventional crystallography, SX does not involve the mounting and alignment of individual crystals in the X-ray beam. Because data from a large number of individual crystals is merged, the radiation dose received by each crystal can be substantially reduced compared to conventional crystallography. Synchrotron SX can also be applied to the study of time-resolved reactions, even down to the millisecond regime, provided a detector with a sufficiently high frame rate is available (e.g., 100 Hz or more). Several serial crystallography experiments have been carried out at the synchrotron using injectors that were initially developed at XFEL sources20,22,23. The two most common types of injector are the Gas Dynamic Virtual Nozzle (GDVN)25 and High Viscous Injector (HVI)9,24,26,27,28. The GDVN is ideal for injecting low viscosity, liquid samples, but requires high flow rates to achieve stable streams, which in turn leads to high sample consumption rates. By contrast, HVI&#8217;s are suitable for high viscosity samples which allows generation of a stable stream at much lower flow rates, leading to much lower sample consumption. The HVI injector, therefore, favors delivery of samples where a viscous carrier is preferable (e.g., lipid-based for membrane proteins) and/or large quantities of sample are not available. SX injectors are generally challenging to use and require extensive training to operate. They also involve lengthy sample transfer protocols, as the sample needs to be loaded into a specialized reservoir, this generally has a high risk associated with it of sample being lost either in the &#8216;dead volume&#8217; or via leakages in the connections. Therefore, it is desirable to optimize the injector design to mitigate any losses prior to the sample reaching the X-ray beam.
Recently, the first SX results were published using Lipidico23&#160;with a lysozyme target,&#160;using an Eiger 16M detector. This injector design limits sample wastage by minimizing the number of steps involved in going from initial crystallization to the transfer of crystals into the injector followed by the delivery of sample to the X-ray beam. This manuscript describes and demonstrates the sample transfer procedure starting from sample preparation, moving on to the injection process, and finally data collection, using the same crystallization vessel. The operation of the injector is also described.

<table>
	<tbody>
		<tr>
			<td>Hen eggwhite lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>Used to grow crystals for testing the injector and the crystals are transferred into silicon grease. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>High vacuum silicon grease</td>
			<td>Dow Corning</td>
			<td>Z273554-1EA</td>
			<td>Used for testing of injector. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>Injector needle (108 &#181;m ID)</td>
			<td>Hamilton</td>
			<td>part No: 7803-05</td>
			<td>www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>Glass gas-tight syringes, 100 &#181;l</td>
			<td>Hamilton</td>
			<td>part no: 7656-01</td>
			<td>Syringes used for sample injection. www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>LCP syringe coupler</td>
			<td>Formulatrix</td>
			<td>209526</td>
			<td>Syringe coupler to mix the samples</td>
		</tr>
		<tr>
			<td>Lipidico injector</td>
			<td>La Trobe Univerity/ANSTO</td>
			<td></td>
			<td>This is a specific piece of equipment that can be accessed through La Trobe University / ANSTO Australian Synchrotron Facility</td>
		</tr>
	</tbody>
</table>

lipidico injection protocol for serial crystallography measurements at the australian synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

Lipidico is an HVI built as an alternative delivery system for use on MX2 (Figure 1). It is ideally suited for SX where crystals are either grown in lipidic cubic phase or transferred to a high viscous inert media.
To demonstrate the injector application silicone grease mixed with lysozyme crystals was used to collect SX data at the MX2 beamline at the Australian synchrotron. To mount the injector on the MX2 beamline the cryogenic nozzle is removed and replaced by...

Watch this Scientific Journal Video about Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron at JoVE.com

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

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.

Research

JoVE Journal

Bioengineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...