We would like to thank Molly S.C. Gravett for helpful discussions and the ABSL facility staff for help with cryo-EM data collection. David P. Klebl&#160;is a PhD student on the Wellcome Trust 4-year PhD program in The Astbury Centre funded by The University of Leeds. The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust (108466/Z/15/Z). This work was funded by a BBSRC grant to Stephen P. Muench&#160;(BB/P026397/1) and supported by research grants to Howard D. White from the American Heart Association (AMR21-236078) and Howard D. White and Vitold Galkin from the U.S. National Institutes of Health (171261).

1. Preparing the system
NOTE: The following protocol describes how to prepare grids of a single sample. Usually, a minimum of 2 replicate grids are prepared for each sample or condition. For faster plunge speeds (less than ~ 20 ms time delay), 3 or 4 replicate grids are typically prepared to account for a reduced number of thin ice areas.
<ol>
	<li>Dilute the protein sample to the target concentration in the desired buffer. Typically, final concentrations &#8805; 2 mg/mL work well for grid p...

<ol>
	<li>Murphy, B. J., 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=Rotary+substates+of+mitochondrial+ATP+synthase+reveal+the+basis+of+flexible+F1-Fo+coupling.">Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling.</a> Science. 364, (2019).</li><li>Benton, D. J., Gamblin, S. J., Rosenthal, P. B., Skehel, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+transitions+in+influenza+haemagglutinin+at+membrane+fusion+pH.">Structural transitions in influenza haemagglutinin at membrane fusion pH.</a> Nature. , 1-4 (2020).</li><li>Dance, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motion+on+ice.">Molecular motion on ice.</a> Nature Methods. , 1-5 (2020).</li><li>D'Imprima, E., 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=Protein+denaturation+at+the+air-water+interface+and+how+to+prevent+it.">Protein denaturation at the air-water interface and how to prevent it.</a> Elife. 8, 42747 (2019).</li><li>Noble, A. J., 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=Reducing+effects+of+particle+adsorption+to+the+air-water+interface+in+cryo-EM.">Reducing effects of particle adsorption to the air-water interface in cryo-EM.</a> Nature Methods. 15, 793-795 (2018).</li><li>Klebl, D. 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=Need+for+speed:+Examining+protein+behaviour+during+cryoEM+grid+preparation+at+different+timescales.">Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales.</a> BioRxiv. , (2020).</li><li>Ravelli, R. B., 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=Cryo-EM+structures+from+sub-nl+volumes+using+pin-printing+and+jet+vitrification.">Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification.</a> Nature Communications. 11, 1-9 (2020).</li><li>Arnold, S. A., 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=Blotting-free+and+lossless+cryo-electron+microscopy+grid+preparation+from+nanoliter-sized+protein+samples+and+single-cell+extracts.">Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.</a> Journal of Structural Biology. 197, 220-226 (2017).</li><li>Razinkov, I., 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=A+new+method+for+vitrifying+samples+for+cryoEM.">A new method for vitrifying samples for cryoEM.</a> Journal of Structural Biology. 195, 190-198 (2016).</li><li>Feng, X., 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=A+fast+and+effective+microfluidic+spraying-plunging+method+for+high-resolution+single-particle+cryo-EM.">A fast and effective microfluidic spraying-plunging method for high-resolution single-particle cryo-EM.</a> Structure. 25, 663-670 (2017).</li><li>Ashtiani, D., 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=Delivery+of+femtolitre+droplets+using+surface+acoustic+wave+based+atomisation+for+cryo-EM+grid+preparation.">Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation.</a> Journal of Structural Biology. 203, 94-101 (2018).</li><li>Rubinstein, J. L., 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=Shake-it-off:+a+simple+ultrasonic+cryo-EM+specimen-preparation+device.">Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.</a> Acta Crystallographica Section D: Structural Biology. 75, (2019).</li><li>M&#228;eots, M. -. E., 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=Modular+microfluidics+enables+kinetic+insight+from+time-resolved+cryo-EM.">Modular microfluidics enables kinetic insight from time-resolved cryo-EM.</a> Nature Communications. 11, 1-14 (2020).</li><li>Kontziampasis, D., 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=A+cryo-EM+grid+preparation+device+for+time-resolved+structural+studies.">A cryo-EM grid preparation device for time-resolved structural studies.</a> IUCrJ. 6, (2019).</li><li>Klebl, D. 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=Sample+deposition+onto+cryo-EM+grids:+from+sprays+to+jets+and+back.">Sample deposition onto cryo-EM grids: from sprays to jets and back.</a> Acta Crystallographica Section D: Structural Biology. 76, (2020).</li><li>White, H., Thirumurugan, K., Walker, M., Trinick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+generation+apparatus+for+time-resolved+electron+cryo-microscopy+using+stepper+motors+and+electrospray.">A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray.</a> Journal of Structural Biology. 144, 246-252 (2003).</li><li>Kaledhonkar, S., Fu, Z., White, H., Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Complex Assembly. , 59-71 (2018).</li><li>Trebbin, 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=Microfluidic+liquid+jet+system+with+compatibility+for+atmospheric+and+high-vacuum+conditions.">Microfluidic liquid jet system with compatibility for atmospheric and high-vacuum conditions.</a> Lab on a Chip. 14, 1733-1745 (2014).</li><li>Klebl, D. P., Sobott, F., White, H. D., Muench, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-grid+and+in-flow+mixing+for+time-resolved+Cryo-EM.">On-grid and in-flow mixing for time-resolved Cryo-EM.</a> Acta Crystallographica Section D: Structural Biology. , (2021).</li><li>He, S., Scheres, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helical+reconstruction+in+RELION.">Helical reconstruction in RELION.</a> Journal of Structural Biology. 198, 163-176 (2017).</li><li>Millar, N. C., Geeves, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limiting+rate+of+the+ATP-mediated+dissociation+of+actin+from+rabbit+skeletal+muscle+myosin+subfragment+1.">The limiting rate of the ATP-mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1.</a> FEBS Letters. 160, 141-148 (1983).</li><li>Kasas, S., Dumas, G., Dietler, G., Catsicas, S., Adrian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+cryoelectron+microscopy+specimens+revealed+by+high-speed+photographic+imaging.">Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging.</a> Journal of Microscopy. 211, 48-53 (2003).</li><li>Glaeser, R. 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=Defocus-dependent+Thon-ring+fading.">Defocus-dependent Thon-ring fading.</a> bioRxiv. , (2020).</li><li>Bagshaw, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beginner's+guide+to+flow+kinetics.">A beginner's guide to flow kinetics.</a> The Biochemist. 42, (2020).</li></ol>

The protocols in this work can be used for fast grid preparation by direct spraying and TrEM experiments. Fast grid preparation can be used to reduce particle interactions with the air water interface5. The main limitations are the available sample concentration and ice thickness on the grid. Within these limits and provided that the sample quality is good, the protocol produces grids suitable for high resolution cryo-EM.
Troubleshooting 
Liquid fl...

Background 
Recent developments in cryo-electron microscopy (cryo-EM) have enabled structural studies of increasingly complex systems at high resolution. With few exceptions, such studies have been limited to biological macromolecules at equilibrium1 or relatively slow reactions2. Many processes in vivo occur on a faster timescale (milliseconds) and there is increasing interest in time-resolved cryo-EM (TrEM) on these timescales3. However, conventional cryo-EM sample preparation by the blotting method is too slow for millisecond TrEM.
The blotting method has other limitations besides poor time resolution. Proteins and protein complexes can suffer from denaturation or preferred orientation on grids4. Reducing the exposure time to the air-water interface during sample preparation has been shown to mitigate preferred orientation and protein denaturation5,6. Thus, fast grid preparation not only enables millisecond TrEM but can also improve grid quality.
Currently, there are three different approaches to automated grid preparation. The first approach uses a pin or capillary that holds a small amount of sample. After establishing contact between the liquid and the grid surface, the sample is &#39;written&#39; onto the grid7,8. The sample application process is relatively slow and takes a few seconds. An alternative approach uses controlled droplet generation by a piezo dispenser and self-wicking grids9. This allows faster dispense to freeze times, but is still limited by droplet and wicking speed (currently reaching 54 ms). The fastest approach so far is the direct spray approach, in which the sample is atomized in a spray nozzle and the small (~ 10 - 20 &#181;m) and fast (&#62; 5 m/s) droplets spread upon contact with the cryo-EM grid. The sample spray can be generated through different ways such as airblast atomizers, surface acoustic waves or ultrasonic humidifiers10,11,12,13. In our experience, the ice thickness with the direct spraying approach is greater but direct spraying enables dispense to freeze times &#60; 10 ms.
This protocol describes step-by-step how a time-resolved EM device (TED) equipped with a microfluidic spray nozzle can be used to prepare grids on a fast timescale14,15. The device has been used to prepare grids with a minimum delay time of 6 ms between sample application and freezing and to rapidly mix and freeze two samples. The design of the TED is based on a previous version16 and is similar to other spray-based time-resolved cryo-EM devices17.
First, the four main parts of the TED setup are described. The core of the TED is the liquid handling unit, which is responsible for sample aspiration and dispensing. A pneumatic plunger moves the grid through the spray into the liquid ethane. Generation of the spray is achieved with microfluidic spray nozzles and freezing is done in a liquid ethane container, which are described briefly. Lastly, the additional features to control the grid environment, especially humidity, are highlighted. This is followed by detailed protocols for the operation of the device and for conducting TrEM experiments. Representative results are given for fast grid preparation and a simple TrEM experiment.
Experimental Setup
The liquid handling unit 
The liquid handling system of the TED is formed by three syringe drive pumps (&#39;pumps 1 - 3&#39;), each equipped with a rotary valve (Figure 1). A power supply provides pumps 1 - 3 with 24 V DC. Communication with the control software (written in Visual Basic and C++) is via a RS232 interface to pump 1. Commands are distributed through the serial I/O expansion ports from pump 1 to pumps 2-3. Pumps 1-3 are equipped with glass syringes (&#39;syringes 1-3&#39;, we use 250 &#181;L/zero dead volume syringes here). Each valve has two positions, &#39;load&#39; and &#39;dispense&#39;. The &#39;load&#39; position is used to aspirate sample into the syringe. A short piece (~ 3 - 4 cm) of 1/16&#34; O.D., 0.01&#8242;&#8242; I.D. FEP tubing is connected via ETFE/ETFE flangeless fittings to the &#39;load&#39; position of valves 1-3. This short piece of tubing reaches into the sample reservoir (typically a 1.5 mL or 0.5 mL plastic tube). The &#39;dispense&#39; position leads to the spray nozzle. Connection between the &#39;dispense&#39; outlet and the spray nozzle is made by PE tubing (~ 20-30 cm length, 0.043&#34; O.D., 0.015&#34; I.D.), with a short piece of sleeve tubing (~ 0.5 cm) and ETFE/ETFE flangeless fittings.
The pneumatic plunger 
The TED uses a pneumatic plunger to accelerate the grid and move it through the sample spray into the liquid ethane container. Negative pressure tweezers hold the grid, screwed into a home-built holder which is mounted to a dual rod pneumatic cylinder (Figure 2A).
Pressure is supplied from a large nitrogen gas cylinder (size W), equipped with a multistage regulator (0 - 10 bar, &#39;main pressure&#39;). Flexible reinforced PVC tubing (12 mm O.D.) connects the regulator to a 12-port manifold where pressurized nitrogen is delivered to the nozzle and the pneumatic plunger. Gas flow through the nozzle is constant, regulated directly at the nitrogen cylinder (main pressure). The connection to the nozzle is made with PU tubing (4 mm O.D., 2.5 mm I.D.), a short piece of PE tubing (~ 8 cm length, 0.043&#34; O.D., 0.015&#34; I.D.) and appropriate connectors. Pressure on the pneumatic plunger is controlled through a solenoid valve. PU tubing (4 mm O.D., 2.5 mm I.D.) connects the solenoid valve with a regulator and the pneumatic plunger, to allow a reduced plunge pressure (&#8804; main pressure). The solenoid valve is computer controlled. A schematic overview of the setup is given in Figure 2B.
Note that with this setup the plunge pressure is always equal or smaller than the spray gas pressure (main pressure). However, the setup can easily be changed by incorporating a second regulator upstream of the spray nozzle to allow higher plunge speeds at low spray gas pressure. High pressures (&#62;&#62; 2 bar) can damage the PDMS spray nozzle.
CAUTION: This is a pressurized system and the &#39;main pressure&#39; should always be &#60; 7 bar.
Pressures between 0.5 and 2 bar are typically used for the pneumatic plunger and show an approximately linear relation between pressure and speed (at the vertical position of the spray). Plunge speeds are measured with an oscilloscope, connected in line with a slide potentiometer (10 k&#937;) and in parallel with a 2 k&#937; resistor (Figure 2C). A power supply provides the potentiometer with 9 V DC. While the approximate plunge speed is set prior to the experiment by setting the plunge pressure, the potentiometer gives a precise readout of the speed after the experiment.
Spray nozzles and liquid ethane container 
The fabrication and operation of gas-dynamic virtual nozzles for spray-based sample delivery has been described elsewhere in detail15. As described above, the &#39;dispense&#39; outlets of valves 1-3 are connected to the liquid inlets of the nozzle (Figure 3A). The pressurized spray gas is connected to the gas inlet of the nozzle. The inlets in the PDMS spray nozzles are such that 0.043&#34; O.D. PE tubing can be used directly without the need for fittings. Our nozzle design contains a &#39;jet-in-jet&#39; geometry for mixing of two samples, similar to the device described in ref.18. A schematic of the design is shown in Figure 3B, a microscopic image of a nozzle is shown in Figure 3C. The layout of the microfluidic device requires the use of three syringes to mix two samples. The spray nozzle is typically positioned at 1-1.5 cm distance from the grid (during sample application).
We use liquid ethane as a cryogen, in a liquid ethane/nitrogen container as used for the standard blotting method. Vertical positioning of the liquid ethane cup is achieved with a laboratory lifting platform.
Control of the spray and grid environment 
The plunger and spray nozzle are contained within a custom built PMMA (acrylic glass) box with a double door (Figure 4A). High relative humidity inside the box is achieved by an air-humidification system at the back of the TED (Figure 4B). Air is supplied by a pump and fed into a first 10&#34; canister (typically used for under sink water purification). The canister is filled with a low (~ 5-10 cm) level of water and also houses a humidifier unit. Mains power to the humidifier is controlled by a digital humidity/temperature controller and a humidity/temperature sensor located inside the acrylic glass box. The controller is set to turn off the pump when the relative humidity reaches &#8805; 90 %. Humidified air from the first canister is pumped through a diffuser, immersed in water in a second 10&#34; canister and then enters the acrylic glass box.
CAUTION: Because the sample is aerosolized in the spray nozzle, hazardous biological or chemical specimen are not suitable as samples.
The run sequence 
The Run Script button in the control software initiates the run sequence. This sequence of commands can be pre-defined in a script file and altered through the software. The most important variables are explained here:
Spray speed: The spray speed determines the liquid flowrate used by the syringe pump. The flowrate can be calculated as follows: The syringe pump motors used here have a fixed step size. The full range of the pump is divided into 48,000 steps. The second important factor is the syringe volume. We typically use 250 &#181;L syringes. The spray speed in the control software is set as number of steps/second. A spray speed of 1000 steps/second corresponds to:
<img alt="Equation 1" src="/files/ftp_upload/62199/62199eq01.jpg" />
Spray volume: The spray volume determines the total volume to be sprayed. Thus, it also determines the duration of the spray. The spray volume in the control software is set as a number of steps. A spray volume of 2000 steps, at a spray speed of 1000 steps/second, leads to a spray duration of 2 s and a total volume of 10.4 &#181;L.
Pre-spray time: This variable defines the time between initiation of the spray and plunge. It is important to choose the delay time such that the spray has sufficient time to stabilize before plunging the grid. Usually, the spray is given 1.5 - 4 s to stabilize before the grid is plunged. The spray is maintained until the grid has moved through. Usually, the liquid flow (and therefore the spray) is stopped 0.5 to 1 s after the grid has been plunged. Using a spray speed of 1000 steps/s and a spray volume of 2000 steps, a typical pre-spray time is 1.5 s, for example.
An exemplary sequence of commands is shown in Figure 5A, the grid position over time is illustrated in Figure 5B.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time resolved device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>acrylic glass box</td>
			<td>USA scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>digital humidity/temperature controller</td>
			<td>THE20 digital humidity/temperature controller</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dual rod pneumatic cylinder</td>
			<td>dual rod pneumatic cylinder TN 10x70</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FEP tubing</td>
			<td>Upchurch Scientific 1/16&#8221; O.D., 0.01&#39;&#39; I.D. FEP tubing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flangeless fittings</td>
			<td>Upchurch Scientific ETFE/ETFE flangeless fittings</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flexible reinforced PVC tubing</td>
			<td>12 mm OD. flexible reinforced PVC tubing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass syringes</td>
			<td>Kloehn 250 &#181;L zero-dead volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>humidifier pump</td>
			<td>Interpret Aqua Air AP3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>liquid ethane container</td>
			<td>from Thermo/FEI VitrobotTM Mark IV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>multistage regulator</td>
			<td>GASARC class 3 multistage regulator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>negative pressure tweezers</td>
			<td>Dumont N5 Inox B negative pressure tweezers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>oscilloscope</td>
			<td>Hantek 6022BE oscilloscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE tubing</td>
			<td>Scientific Commodities Inc. 0.043&#8221; O.D., 0.015&#8221; I.D. PE tubing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>power supply</td>
			<td>Mean Well GSM160A24-R7B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>power supply</td>
			<td>Wanptek KPS305D power supply</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PU tubing</td>
			<td>SMC TU0425 4 mm O.D., 2.5 mm I.D. PU tubing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>regulator</td>
			<td>Norgren R72G-2GK-RMN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>slide potentiometer</td>
			<td>PS100 slide potentiometer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>solenoid valve</td>
			<td>SMC NVJ314M solenoid valve</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>syringe drive pumps</td>
			<td>Kloehn V6 48K model</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents &#38; Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>apoferritin from equine spleen</td>
			<td>Sigma-Aldrich, A3660</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, A2383</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cryo-EM grids</td>
			<td>Quantifoil 300 mesh Cu, R 1.2/1.3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich E3889</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F-actin</td>
			<td>Provided by H.D. White (for preparation procedure, see ref. 1)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glow-discharger</td>
			<td>Cressington 208 carbon coater with a glow-discharge unit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich, H7006</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KAc</td>
			<td>Sigma-Aldrich, P1190</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich, M8266</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich, M1254</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, S9888</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skeletal muscle myosin S1</td>
			<td>Provided by H.D. White (for preparation procedure, see ref. 2)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ref 1</td>
			<td>Spudich, J. A. &#38; Watt, S. The regulation of rabbit skeletal muscle contraction I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. Journal of biological chemistry 246, 4866-4871 (1971).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ref 2</td>
			<td>White, H. &#38; Taylor, E. Energetics and mechanism of actomyosin adenosine triphosphatase. Biochemistry 15, 5818-5826 (1976).</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fast grid preparation for time-resolved cryo-electron microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Fast grid preparation with the TED 
As a test specimen for fast grid preparation, we have used apoferritin from equine spleen at 20 &#181;M in 30 mM HEPES, 150 mM NaCl, pH 7.5. A reconstruction at 3.5 &#197; resolution was obtained from 690 micrographs as described in ref.15 (Figure 7A). The defocus range was chosen so that particles can easily be identified in the raw images (Figure 7B). A typical grid prepared with...

Watch this Scientific Journal Video about Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy at JoVE.com

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Hello, and welcome to this JoVE"video. We want to introduce you to the setup that we've got here for rapid grid making, a time-resolved study that we're developing over in Leeds. Be aware this isn't a standard system, but we hope that by giving you a detailed account of all the things and the parameters that we do, you'll be able to develop similar systems in your own laboratories.A detailed description of the device is given in the Richmond protocol. Here, we're going to give a step-by-step guide for fast grid preparation and time-resolved EM.In our experience, protein concentration should be two milligrams per milliliter or higher. The protocol requires a minimum volume of about 50 microliters.Turn on the device. Then turn on the control PC and start the control software. Initialize all syringe pumps by pressing the initialize button on each syringe pump.Turn on the potentiometer power supply, set it to nine volts, and start the oscilloscope control software. Pressurized nitrogen gas is needed to generate the spray. Open the nitrogen cylinder and set the pressure to 2 bar.Ensure all syringe pump valves are in load position. To do this, switch all valves to dispense in the control software and then back to load. Set all syringes to zero.Any air bubbles present in the system should be removed at this stage. To do this the syringes may have to be unscrewed, bubbles removed manually, and the syringes mounted again when containing no more bubbles. The liquid handling system is usually stored in water.Before loading the sample, the system is equilibrated with buffer. This is done by washing the tubing with an excess of buffer. Place a tube containing at least 200 microliters of buffer onto syringe one.The top of the tube must be pierced to attach it. Aspirate a suitable amount of buffer, between 50 and a hundred microliters with syringe one using the control software. Switch valve one to dispense.Dispense all of the liquid in syringe one. Return valve one to the load position, and press the initialize button on syringe one to prepare the system for the next wash cycle. These steps are usually repeated three times to ensure thorough washing of the tubing.Next, the spray nozzle is positioned. Place an EM grid in the plunging arm. Position the grid in front of the spray nozzle.Dispense buffer using the control software. If nozzle and grid are aligned, liquid should accumulate after spraying for an extended period of time. If necessary, adjust the nozzle position and then check again where the liquid accumulates on the grid.Next, adjust the position of the ethane cup. The tip of the tweezers should reach approximately to the center of the ethane cup. Perform a test run to ensure all settings are correct.Close the humidity chamber. Make sure the path of the plunger is clear. Aspirate the volume of buffer required for a single run into syringe one.Switch valve one to dispense. Start the run by pressing run'in the control software. Check that syringe pump one is moving, two seconds in this case, and the grid is plunged after an appropriate delay, 1.5 seconds here.When the run is finished, set the pressure on the plunger arm to the desired value. Press OK'in the control software to release the pressure from the plunging arm. Now the ethane cup is cool and filled with liquid ethane.Glow-discharge the grids before use. A typical glow-discharge is 90 seconds at 10 milliamps in 0.1 millibar air. The tubing is then equilibrated with sample.If the available sample volume is low, the tubing may be equilibrated with only one dead-volume. Aspirate sample. Typically the dead volume is around 30 microliters.Switch valve one to dispense. Dispense the sample through tubing and nozzle. Then aspirate the amount of sample required for a single run.Switch valve one to dispense. Check that the relative humidity has reached the desired value. We typically prepare grids at 60%or higher.This can take a few minutes. Place the tweezers holding a glow-discharged grid in the plunging arm. Place the potentiometer slide in the start position, ready for speed measurement.Set the trigger for the speed measurement in the oscilloscope software. Place the liquid ethane cup, and close to humidity chamber. Make sure the path of the plunger is clear.Start the run in the control software. When the run is finished, press OK'in the control software to release pressure from the plunging arm. Open the humidity chamber, loosen the connection between plunging arm and tweezers.Move up the plunging arm while keeping the grid in liquid ethane. Then transfer the grids to liquid nitrogen and into storage. Save the oscilloscope measurement.Manually reset the position of the potentiometer slider and plunger. Repeat the protocol to prepare replicate grids. Time-resolved experiments are done in a similar way.Higher stock concentrations are needed for time-resolved experiments because of dilution in the mixing step. For a time-resolved experiment, use all three syringes. Tubing is attached to the syringe pumps and to the spray nozzle.The microfluidic spray nozzle used here also contains a mixing element. Note that longer tubes will give a larger dead-volume and washing equilibration steps require more buffer and sample. Equilibrate all syringes with buffer and sample separately.Typically, syringe one is used for sample A'and syringes two and three are used for sample B'After the tubing is equilibrated, load sample A into syringe one and sample B into syringes two and three. Then, switch valves one to three to dispense. Start the run in the control software.Note that a different run script is needed for a time-resolved experiment. Different time delays can be achieved in two ways. By adjusting the plunger speed, the time delay from mixing to freezing can be changed.A faster plunge speed will lead to a shorter time delay. Alternatively, the spray ethane distance is changed by adjusting the vertical position of the spray nozzle. Increasing the distance will result in a longer time delay.In the electron microscope at low magnification, a typical grid looks like this. Areas of thin ice, as indicated, are suitable for data acquisition. At higher magnifications, particles should be clearly visible.With a test specimen such as apoferritin, a relatively small data set from a single grid is sufficient for a reconstruction at three to four angstrom resolution. For time-resolved experiments, we collect data sets from different time points or grids. It's useful to combine the data for 3D classification and then trace particles back to their time points.This way the data can provide structural information and information on the reaction kinetics. In conclusion, we hope you enjoyed the video. We hope that with the detailed descriptions in the JoVE paper along with the video, you'll be able to conduct your own experiments via rapid grid making, which has been shown to alleviate some of the problems from mutation or to help with interactions at the air-water interface, all you need for conducting your own time-resolved studies to try and trap some of those non equilibrium states.So, happy grid making.

fast-grid-preparation-for-time-resolved-cryo-electron-microscopy

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Biochemistry

3.8K ビュー.  University of Leeds. こんにちは、このJoVE"ビデオへようこそ。リーズで開発中の時間を取り上げた研究である、迅速なグリッド作成のためにここに来たセットアップをご紹介します。これは標準システムではありませんが、私たちが行うすべてのこととパラメータの詳細な説明を提供することで、自分の研究室で同様のシステムを開発できることを願っています。デバイスの詳細な説?...