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 preparation with the TED. Note that the sample can be kept on ice until step 10, from step 10 onwards the sample will spend considerable time at room temperature as it takes approximately 20 min to prepare 3-4 grids.</li>
	<li>Turn on the TED. Then turn on the control PC and start the control software.</li>
	<li>Initialize all syringe pumps by pressing the Initialize button (lower black button) on each syringe pump.</li>
	<li>Turn on the potentiometer power supply, set it to 9 V and start the oscilloscope control software.</li>
	<li>Ensure that the regulator valve of the N2-cylinder is closed. Open the cylinder valve. Then, slowly open the regulator valve setting the outlet pressure to the desired value for the spray gas pressure (typically 1-2 bar). For the PDMS nozzles used here, do not use pressures higher than ~ 2.5 bar to avoid damage to the nozzle. The continuously flowing gas prevents liquid from dripping and accumulating at the nozzle tip which can lead to irreproducible spraying.</li>
	<li>Ensure all syringe pump valves are in the &#39;load&#39; position. This can be done by switching all valves to the &#39;dispense&#39; position in the control software and then back to &#39;load&#39;. Leave all valves switched to &#39;load&#39;. Set all syringes to zero.</li>
	<li>Remove any air-bubbles present in the system 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.</li>
	<li>The liquid handling components are usually stored in H2O. Before loading the sample solution, equilibrate the liquid system with buffer by washing the tubing with an excess of buffer. Use an appropriate maximum liquid flowrate to avoid overpressure on the spray nozzle. Liquid flowrates &#62; 10 &#181;L/s may cause damage to the PDMS nozzles described here.</li>
	<li>Place a 1.5 mL tube containing &#8805; 200 &#181;L buffer onto syringe 1 (the top must be pierced to attach to tubing).
	<ol>
		<li>Make sure valve 1 is in &#39;load&#39; position. Switch in the control software, if necessary.</li>
		<li>Aspirate the desired amount (typically 50-100 &#181;L) of buffer with syringe 1, through the control software.</li>
		<li>Switch valve 1 to the &#39;dispense&#39; position, through the control software.</li>
		<li>Dispense all of the liquid in syringe 1, through the control software.</li>
		<li>Return valve to &#39;load&#39;, reset the syringe position to &#39;0&#39; in the program and press initialize on syringe 1, to prepare the system for the next cycle. 
		NOTE: Steps 1.9.1 - 1.9.5 are usually repeated three times to ensure thorough washing of the tubing.</li>
	</ol>
	</li>
	<li>Load tweezers with an EM grid (no requirement for any specific type) by loosening the plunging arm clamp, placing the tweezers in the clamp and tightening the clamp.</li>
	<li>Manually move the plunging arm so that grid and nozzle are at the same height (the &#39;sample application&#39; position). If nozzle and grid are aligned, liquid will accumulate on the grid after spraying for an extended&#160;period. If necessary, adjust the nozzle position.</li>
	<li>Adjust the position of the liquid ethane cup by manually moving the plunging arm with mounted tweezers to its end position (reaching into the ethane cup). When set up correctly, a grid held by the tweezers will reach approximately the center of the liquid ethane cup.</li>
	<li>Check that the &#39;run script&#39; will use the desired flowrates, volumes and timings. See section &#39;The run sequence&#39; above for details.</li>
	<li>Make sure nothing is obstructing the path of the plunger. Aspirate the volume of buffer required for a single run into syringe 1. This is done in the control software, see section &#39;The run sequence&#39; for typical setting and volumes. Then make sure valve 1 is switched to the &#39;dispense&#39; position. Perform a test run by pressing Run Script in the control software. 
	CAUTION: Stay clear of the TED until the run sequence is finished. Moving parts could cause injury!</li>
	<li>When the &#39;run sequence&#39; is finished, set the pressure on the plunging arm to the desired value (typically 1-2 bar). Only then press OK in the control software to release pressure from the plunging arm. If the &#39;run sequence&#39; or pressure on the plunging arm need to be adjusted, these settings may be changed at this stage and steps 1.13 - 1.15 repeated.</li>
</ol>
2. Fast Grid Preparation
<ol>
	<li>Fill the liquid ethane/nitrogen container first with liquid nitrogen. When sufficiently cold and free of liquid nitrogen, fill the cup with liquid ethane. Avoid solidification of the liquid ethane. This step is the same as for conventional grid preparation. 
	NOTE: To minimize ethane contamination, ensure that steps 2.2 - 2.13 are performed as quickly as possible 
	CAUTION: Liquid ethane is a cryogen and flammable. Care should be taken when handling.</li>
	<li>Prepare cryo-EM grids for glow-discharge. We typically use holey carbon grids and glow-discharge for 90 s, at 0.1 mbar air pressure and 10 mA. Usually, no more than 4 grids are glow discharged at a time. The grids are used within 30 min of glow-discharging.</li>
	<li>Equilibrate the tubing with sample (following steps 1.9.1 - 1.9.5, using sample instead of buffer). If the available sample volume is low, the tubing may be equilibrated with only 1 dead volume.</li>
	<li>Aspirate the amount of sample that is needed for a single run into syringe 1 in the control software (see section &#39;The run sequence&#39; for details). Then switch valve 1 to the &#39;dispense&#39; position.</li>
	<li>Check that the relative humidity has reached the desired value (we typically prepare grids at 60 % relative humidity or higher). Once &#8805; 60 % humidity is reached, open the humidity chamber only for a minimal amount of time, to maintain high humidity.</li>
	<li>Place the tweezers, holding a glow-discharged grid, in the pneumatic plunger and fix them. Move the plunger to its start position (at the top).</li>
	<li>Make sure the slider of the potentiometer is in the start position, ready for the measurement, by moving it manually to contact the plunger. Set the trigger on the oscilloscope in the oscilloscope software.</li>
	<li>Place the liquid ethane/nitrogen container.</li>
	<li>Press Run Script in the control software. When prompted, click OK in the control software to start the run. 
	CAUTION: Stay clear of the TED until the run sequence is finished. Moving parts could cause injury!</li>
	<li>Once the &#39;run sequence&#39; is completed, release the pressure on the pneumatic plunger by clicking OK in the control software.</li>
	<li>Open humidity chamber, loosen the connection between plunging arm and tweezers with one hand while securing the tweezers with the other. When the tweezers are free, move the plunging arm up while keeping the grid in the liquid ethane. Then transfer the grid to its storage space in the surrounding liquid N2. After freezing, the grid needs to be kept at liquid N2 temperature at all times.</li>
	<li>Save the oscilloscope measurement. Manually reset the position of the potentiometer slider and plunger afterwards.</li>
	<li>Repeat steps 2.4-2.12 to prepare replicate grids</li>
	<li>Transfer the grids into long-term storage until grid clipping and data collection.</li>
	<li>Wash the system with buffer, according to steps 1.9.1 - 1.9.5. Then wash the system with H2O, according to steps 1.9.1 - 1.9.5.</li>
	<li>Turn off the main nitrogen gas regulator and turn off the power.</li>
	<li>Place the ethane/nitrogen container in a fume hood to let it warm up and let the liquid ethane and N2 evaporate.</li>
</ol>
3. Time-resolved cryo-EM
NOTE: When time-resolved experiments are conducted with the TED, there are additional aspects to be considered, although the basic setup and variables remain the same. It is assumed here that two solutions are mixed in a 1:1 (v/v) ratio to produce the final mixture which is deposited on the grid. Follow the protocol described in &#39;1. Fast grid preparation with the TED&#39;, with the following changes:
<ol>
	<li>Use higher stock concentrations for mixing experiments than for simple spray experiments. Mixing in a 1:1 (v/v) ratio will result in a 2x dilution of each component.</li>
	<li>For a rapid mixing experiment, use all three syringes rather than just a single one.
	<ol>
		<li>Attach tubing to syringe pumps 2-3.</li>
		<li>Attach tubing from syringe pumps 2-3 to the spray nozzle.</li>
		<li>Equilibrate all three syringes in buffer and sample separately. Typically, syringe 1 is filled with sample A and syringes 2-3 are filled with sample B (Figure 3).</li>
	</ol>
	</li>
	<li>Change the run sequence. An example for a run sequence using all 3 syringes for a rapid mixing experiment is given in Figure 6. See also section &#39;The run sequence&#39; for details.</li>
	<li>Different time delays can be achieved in two ways:
	<ol>
		<li>Change the plunger speed. By adjusting the plunger speed, the time delay can be changed in a relatively narrow range. For example, with a spray/ethane distance of 2 cm, the plunger can be moved at 1 m/s or 2 m/s to give a time delay of 20 ms or 10 ms, respectively. This is done as described in step 1.15.</li>
		<li>Change the spray/ethane position by adjusting the (vertical) position of the spray nozzle. If the nozzle is positioned at a 5 cm distance from the ethane surface, for example, a plunge speed of 1 m/s gives a time delay of 50 ms. Achieving significantly longer time delays requires further modifications to the setup. 
		NOTE: Due to laminar flow in the flow focussing region of the nozzle, we do not expect significant mixing in this part of the nozzle. Instead, we expect that mixing occurs during spray generation, in droplets en route to the grid and during droplet spreading on the grid. The time of flight for spray droplets to reach the grid is estimated &#8804; 1 ms (for a droplet speed of &#8805; 10 m/s and a nozzle-grid distance of 1 cm). Thus, only the time between droplet landing and vitrification is considered the &#39;time delay&#39;.</li>
	</ol>
	</li>
</ol>

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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. 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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 flowrate and grid speed determine the amount of liquid deposited on the grid. With the example settings given above (liquid flowrate 5 &#181;L/s and plunge speed 1-2 m/s), a good coverage of the grid with droplets is expected. If the nozzle was not aligned well (so spray droplets miss the grid), frozen grids show very few or no droplets.
Contamination of grids cannot be entirely avoided (see Figure 7C) but can be reduced by minimizing exposure time of liquid ethane to a humid environment. This is achieved by preparing replicate grids as quickly as possible (&#8804; 20 min for 4 grids). We typically prepare 4 grids before replacing the liquid ethane.
Vitreous ice on grids may (partially) crystallize if the initial cooling in liquid ethane is too slow. In agreement with a previous study22, we have found low plunge speeds (&#60; 0.5 m/s) lead to an increase of crystalline ice. Areas of very thick ice (&#8805; 200 nm) typically show crystalline ice due to inherently slower cooling. If the grid warms up during any of the steps following grid preparation (transfer from liquid ethane to liquid nitrogen, transfer to storage, etc.) crystalline ice may occur, too.
Data acquisition for TED prepared grids 
The average ice thickness produced by the TED is thicker than the &#34;ideal&#34; cryo-EM grid prepared on a standard blotting system. Ice thickness can also vary between acquisition areas. This means that acquisition areas need to be selected carefully to give the thinnest possible ice. The defocus range may need to be adjusted to obtain high contrast images. When the ice is thick and particles have low contrast, we suggest an initial pilot data acquisition using very high defocus values (3 - 5 &#181;m). Recent work suggests that high resolution can still be achieved using such high defocus values23.
For conventional cryo-EM data collection, an entire dataset is often collected from a single grid. However, we have found the collection of multiple small datasets and merging of these datasets useful. This way, multiple timepoints can be recorded within limited microscope time and timepoints of interest can be identified.
Processing and analysis of TrEM data 
Merging of datasets can routinely be done in most common cryo-EM data processing software. As described above and in the work of others, merging of datasets from separate timepoints can be useful17. It is important that particles can be traced back to their original subset (timepoint) throughout the processing.
Conventional kinetic measurements (for example using light scattering or fluorescence) can be a valuable addition to TrEM experiments. Rate constants from biochemical measurements can be used to predict lifetimes of the intermediate state of interest, or in confirming the kinetics observed by TrEM. For a basic introduction to reaction kinetics and their relation to time-resolved structural studies, see ref.24.
There is a number of possible reasons for differences between TrEM and conventional kinetic experiments. As shown in Figure 8D, TrEM data can show significant variations per-micrograph. We also note that preliminary data suggests at least 5% variability in relative particle numbers between replicate grids. Particle classification (class assignment) could be imperfect, particularly if states are structurally similar or if a structure is highly flexible. Particles, and thus TrEM derived kinetics, might also be influenced by particle interactions with the air-water interface because such interactions are reduced but not eliminated at fast speeds.
This work provides some guidelines for TrEM but we note that this is an active area of research, and we expect further advances in the future. While TrEM experiments require significant experimental effort, they can offer high resolution insight into the dynamics of macromolecular complexes.

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 a time delay of 10-40 ms shows sufficient areas of thin ice (Figure 7C) to allow for collection of &#62; 1000 micrographs. The resulting resolution is likely limited by ice thickness, tomographic analysis of a number of different grids showed 96 &#177; 33 nm ice thickness6.
Time-resolved cryo-EM 
In order to demonstrate rapid mixing and time-resolution using the TED, we have used dissociation of the actomyosin complex by mixing with ATP as a model reaction19. This choice was based on the easy distinction between actomyosin and free F-actin from raw micrographs and the well understood kinetics of the reaction.
In the following, the representative results for TrEM of dissociation of the actomyosin complex by ATP are shown. Detailed experimental procedures and results are provided in ref.19. In brief, F-actin and rabbit skeletal myosin S1 were mixed in 10 mM MOPS, 50 mM KAc, 2 mM MgCl2, 0.1 mM EGTA, pH 7 at final monomer concentrations of 40 &#181;M to prepare the actomyosin (AM) complex. AM complex was loaded into syringe 1 and 200 &#181;M MgATP in the same buffer was loaded into syringes 2 and 3. The key experimental parameters for the two different timepoints prepared are listed in Table 1.
For both timepoints,&#160;the result was a 1:1 v/v mixture of AM complex and MgATP, giving final concentrations of 20 &#181;M AM complex and 100 &#181;M MgATP. Calculated mixing to freezing times were 7 and 13 ms as shown in Table 1. The estimated time of flight for the spray droplets between nozzle and grid was &#60; 1 ms.
A small cryo-EM dataset (306 and 123 micrographs) was acquired for each timepoint and the combined data processed using standard helical processing procedures20. The resulting consensus reconstruction (both timepoints combined) is shown in Figure 8A. The combined data was then subjected to focussed classification and split into an AM-complex and an F-actin class (Figure 8B, C). Then, the fraction of AM-complex or F-actin particles for each timepoint was calculated and is shown in Figure 8D.
The second order rate constant for AM-complex dissociation is 1.5 &#183; 106 M-1 s-1&#160;21. Under the assumption that
<img alt="Equation 2" src="/files/ftp_upload/62199/62199eq02.jpg" />
the integrated rate law can be approximated to:
<img alt="Equation 3" src="/files/ftp_upload/62199/62199eq03.jpg" />
Where [AM-complex] is the time-dependent concentration of AM-complex, [AM-complex]0 is the initial concentration, k is the second order rate constant and [ATP]0 is the (initial) ATP concentration.
Using this equation, the kinetics of AM-complex dissociation were modelled. The model agrees reasonably well with the experimental TrEM data. There is significant per-micrograph variation as shown in Figure 8D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62199/62199fig01.jpg" /> 
Figure 1: The time-resolved EM device (TED). (A) Overview of the TED. (B) The liquid handling unit with pumps 1-3. The fourth pump on the left is not used in this work. Load and dispense positions are indicated for valve 1, as is the sample reservoir (&#39;sample 1&#39;). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62199/62199fig02.jpg" /> 
Figure 2: Pneumatic plunger and linear potentiometer. (A) Details of the pneumatic plunger of the TED with important components labelled. (B) Schematic of the pneumatic system of the TED. Numbers correspond to the N2 cylinder (1), the main pressure regulator (2), the spray nozzle (3), the solenoid valve (4), the plunge speed regulator (5) and the dual rod pneumatic cylinder (6). (C) Schematic of the circuit for the potentiometer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62199/62199fig03.jpg" /> 
Figure 3: Gas-dynamic virtual nozzles for mixing and spraying. (A) A nozzle with 4 inlet tubes connected, to supply to samples (A and B) and N2 gas. (B) Schematic of the central section of the PDMS spray nozzles with double flow focussing geometry to achieve flow focussing (for downstream mixing) and gas focussing or atomization. Sample B is shown in yellow and sample A in purple. (C) Microscopic image of the mixing and atomization sections of the microfluidic nozzle. Image from ref.15. Scale bar 100 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62199/62199fig04.jpg" /> 
Figure 4: Environmental control of grid and spray in the acrylic glass box. (A) View from the front and (B) from the side of the TED. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62199/62199fig05.jpg" /> 
Figure 5: The run sequence. (A) An example run sequence showing variables at top and the sequence of commands at the bottom. Some of the key commands are annotated in red. (B) Grid position over the time course of a &#39;run sequence&#39; shown as a black solid line. The start position of the grid is at 0 cm. Spray is initiated at t = 0 s for 2 s, the distance between spray and liquid ethane is ~ 2 cm. At 1.5 s, the grid starts moving with 2 m/s, through the spray and into the liquid ethane. The sequence ends as the spray stops after 2 s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62199/62199fig06.jpg" /> 
Figure 6: Example run sequence for a rapid mixing experiment. Important differences to Figure 5 A are annotated in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62199/62199fig07.jpg" /> 
Figure 7: Representative results from a simple spray experiment. (A) 3.5 &#197; reconstruction of equine apoferritin from a grid prepared in 36 ms between spraying and vitrification (EMD- 10533). (B) Raw image of the same apoferritin sample. (C) Low magnification view of the grid, one exemplary grid-square containing thin ice is highlighted by the dashed blue rectangle, the area of thin ice within this grid-square is indicated by the blue arrow. Areas of thick ice or contamination are indicated with asterisks. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62199/62199fig08.jpg" /> 
Figure 8: Representative results from a mixing experiment. (A) Consensus reconstruction of the AM-complex shown at a high threshold (coloured) and low threshold (transparent). One myosin-density is labelled and highlighted with a dashed line. The reconstruction was generated from data from both timepoints combined. (B) F-actin class showing no myosin bound. (C) AM-complex class showing myosin bound (myosin in grey, bound primarily to the light red actin subunit). (D) Comparison between kinetic model and experimental TrEM data. Shown is the fraction of AM-complex relative to the initial concentration. Large solid black dots indicate averaged TrEM data, small dots show per-micrograph TrEM data. The purple dashed line represents the kinetic model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62199/62199fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">flowrate (&#181;L/s)</td>
			<td rowspan="3">spray/ethane distance (cm)</td>
			<td rowspan="3">plunge speed (m/s)</td>
			<td rowspan="3">time-delay (ms)</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td>syringe 1</td>
			<td>syringe 2</td>
			<td>syringe 3</td>
		</tr>
		<tr>
			<td>(AM)</td>
			<td>(ATP)</td>
			<td>(ATP)</td>
		</tr>
		<tr>
			<td>7 ms</td>
			<td>2.08</td>
			<td>1.04</td>
			<td>1.04</td>
			<td>1.4</td>
			<td>2</td>
			<td>7</td>
		</tr>
		<tr>
			<td>13 ms</td>
			<td>1.04</td>
			<td>0.52</td>
			<td>0.52</td>
			<td>2</td>
			<td>1.6</td>
			<td>13</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental settings for TrEM of AM complex dissociation by ATP

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 ...

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

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Biochemistry

3.9K Views.  University of Leeds. 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.

Video: Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...