We acknowledge Gebhard Schertler, Rafael Abela and Chris Milne for supporting the use of high viscosity injectors at the PSI. Richard Neutze and his team are acknowledged for discussions on time-resolved crystallography and sample delivery using high viscosity injectors. For financial support, we acknowledge the Swiss National Science Foundation for grants 31003A_141235, 31003A_159558 (to J.S.) and PZ00P3_174169 (to P.N.). This project has received funding from the European Union&#39;s Horizon 2020 research and innovation program under the Marie-Sklodowska-Curie grant agreement No 701646.

1. Protein Crystal Sample Preparation
<ol>
	<li>About 30 min before the sample is to be injected, load 50 &#181;L of crystal-laden monoolein based LCP in a 100 &#181;L syringe.</li>
	<li>For injection at atmospheric pressure: Load 10 &#181;L of liquid paraffin into the back of a second syringe. Holding the syringe vertically, expel the air bubbles from the syringe.
	<ol>
		<li>For injection into vacuum environment: Load 5 &#181;L of MAG 7.9 and 5 &#181;L of liquid paraffin into the back of a second syringe. Holding the...

The TR-SFX method with the viscous extrusion injector has proven to be a viable technique for structural dynamics studies of bacteriorhodopsin9,10 and photosystem II13 and now seems ready to study proteins driving other photo biological processes such as light-driven ion transport or sensory perception5,50. The protocols described above were designed to maximize the success of TR-S...

Serial femtosecond crystallography (SFX) is a structural biology technique that exploits the unique properties of X-ray free electron lasers (XFEL) to determine room temperature structures from thousands of micrometer-sized crystals while outrunning most of the radiation damage by the &#34;diffraction before destruction&#34; principle1,2,3.
In a time-resolved extension of SFX (TR-SFX), the femtosecond pulses from the XFEL are used to study structural changes in proteins4,5. The protein of interest is activated with an optical laser (or another activity trigger) just prior to being shot by the XFEL in a pump-probe setup. By precisely controlling the delay between pump and probe pulses, the target protein can be captured in different states. Molecular movies of structural changes over eleven orders of magnitude in time demonstrate the power of the new XFEL sources to study the dynamics of several protein targets6,7,8,9,10,11,12,13. Principally, the method joins the dynamic spectroscopic and static structural techniques into one, providing a glimpse into protein dynamics at near atomic resolution.
Simple systems for TR-SFX may contain an endogenous trigger of activation with a photo-sensitive component like retinal in bacteriorhodopsin (bR)9,10, the chromophores in photosystem II12,13, photoactive yellow protein (PYP)6,7 reversibly photoswitchable fluorescent protein11, or a photolyzable carbon monoxide in myoglobin8. Exciting variations of the technique still in development rely on mix and inject schemes14,15 to study enzymatic reactions or an electric field used to induce structural changes16. Given that XFEL sources have only been available for a few years and extrapolating past successes into the future, the method shows potential as a real game-changer with respect to our understanding of how proteins function.
Because biological samples are destroyed by a single exposure to a high power XFEL pulse, new approaches to protein crystallography were necessary. Among these procedures, the ability to grow large amounts of uniform microcrystals needed to be developed17,18,19. To enable data collection at an XFEL, these crystals must be delivered, discarded, and then renewed for each XFEL pulse. Given that XFELs fire usable pulses at 10-120 Hz, sample delivery must be fast, stable, and reliable, while also keeping the crystals intact and limiting consumption. Among the most successful solutions is a high viscosity micro-extrusion injector, which delivers a continiously streaming column of room temperature crystal-laden lipidic cubic phase (LCP) across the pulsed X-ray beam20. Randomly oriented crystals, embedded in the LCP stream, that are intercepted by the XFEL pulses scatter X-rays onto a detector where a diffraction pattern is recorded. LCP was a natural choice for a sample delivery medium as it is frequently used as a growth medium for membrane protein crystals17,21,22,23, yet other high viscosity carrier media24,25,26,27,28,29,30 and soluble proteins31 have also been used in the injector. SFX with the high viscosity injector has been successful during the structure determination of membrane proteins13,32 including G protein-coupled receptors (GPCRs)33,34,35,36,37, with data quality sufficient for native phasing38,39 while being both time and sample efficient. Currently, these injectors are being used more routinely for room temperature measurements at synchrotron sources28,30,40,41 as well as during the more technically demanding TR-SFX experiments at XFELs9,10,13,42.
Comparable TR-SFX experiments have been carried out using other injector types like liquid phase delivery in a flow focused nozzle6,7,12, however, this method requires protein amounts not available for many biologically interesting targets. For the determination of static structures using viscous extrusion an average consumption of 0.072 mg of protein per 10,000 indexed diffraction patterns in comparison to 9.35 mg for the liquid jet nozzles have been reported (i.e., about 130 times more sample efficient)20. The high viscosity injector has been shown to be a viable sample delivery device for TR-SFX while only sacrificing some of this sample efficiency43. In Nogly et al. (2018)10, for example, sample consumption was about 1.5 mg per 10,000 indexed patterns, which compares favorably to similar TR-SFX experiments using the PYP where average sample consumption was much higher with 74 mg of protein per 10,000 indexed patterns6. High viscosity injectors thus have clear advantages when the amount of protein available is limiting or when crystals are grown directly in LCP.
For TR-SFX using high viscosity injectors to yield the most reliable data several technical issues need to be addressed: the flow speed needs to remain above a minimum critical value; the hit-rate should be maintained at a level that does not render data collection slow (e.g., greater than 5%); and sample has to be delivered without excessive disruptions. Ideally, these conditions are already met long before a scheduled TR-SFX experiment to use available XFEL time as efficiently as possible. Pricipally, a slowdown in the LCP stream may allow probing crystals that were activated with more than one optical laser pulse and result in mixed active states, or probing pumped material when umpumped material is expected in the beam. An additional benefit of injection pre-testing is that downtime during data collection at an XFEL is minimized as time relegated to replacing clogged nozzles, changing out non-extruding samples, and other maintenance tasks is reduced.
Here, we present a method to optimize sample delivery for TR-SFX data collection with a high viscosity micro-extrusion injector. For simplicity, the described methods do not rely on access to an X-ray source, although work at a synchrotron beamline29 would provide further information on expected hit rates and crystal diffraction. Our protocols were developed to optimize experiments to capture retinal isomerization in the proton-pump bacteriorhodopsin10 and are carried out in two phases starting with preparing crystal samples for extrusion followed by monitoring the extrusion using a high-speed camera setup. In phase one, the crystal-laden LCP is mixed with additional LCP, low transition temperature lipids, or other additives to ensure the final mixture is suitable for delivery into the sample environment without clogging or slowing. A new three-way syringe coupler was developed to improve mixing performance and sample homogeneity. The second phase consists of an extrusion test recorded by a high-speed camera to directly measure the extrusion speed stability. Following the analysis of the video data, adjustments can be made to the sample preparation protocol to improve experimental outcomes. These procedures can be adapted to prepare other proteins for TR-SFX data collection, with minimal modifications, and will contribute to the efficient use of limited XFEL beamtime. With new XFEL facilities just starting their operation44,45 and the transfer of injector-based serial data collection methods to synchrotrons28,30,40,41, the next few years will surely continue to provide exciting new insights into the structural dynamics of an ever-wider range of protein targets.

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			<td>Purchasable from Uwe Weierstall (weier@asu.edu)</td>
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improving high viscosity extrusion of microcrystals for time-resolved serial femtosecond crystallography at x-ray lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The ideal starting material for the procedures described here (Figure 3) are high densities of microcrystals incorporated into viscous carrier medium for the injector. The procedure calls for about 50 &#181;L of crystal laden carrier for each preparation. These can be grown directly in LCP as with the bR9,10 used here, as an example (Figure 4), or prepared using crystals ...

Watch this Scientific Journal Video about Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers at JoVE.com

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

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

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.

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8.9K visualizações.  Paul Scherrer Institut. Este protocolo foi projetado para garantir que os problemas frequentemente encontrados durante experimentos cristalográficos em série resolvidos no tempo não comprometam a qualidade dos dados. Uma grande vantagem deste método é sua adaptabilidade aos sistemas de cristal individuais. O teste de extrusão da câmera de alta velocidade também nos permite medir diretamente a estabilidade da extrusão.Primeiro, carregue 50 microliters de monooleína cristalina baseada lCP em uma serin...