We thank the Herzik lab members for critically thinking and providing feedback on this manuscript and the video content. M.A.H.Jr. is supported by NIH R35 GM138206 and as a Searle Scholar. H.P.M.N is supported by the Molecular Biophysics Training Grant (NIH T32 GM008326). We would also like to thank Bill Anderson, Charles Bowman, and Dr. Gabriel Lander at the Scripps Research Institute for help designing, assembling, and testing the manual plunger shown in the video.

1. Prepare the manual plunging environment
NOTE: Estimated operating time: 5-30 minutes
<ol>
	<li>Locate the manual plunger in a 4 &#176;C cold room where a humidifier can be co-located to maintain the room close to 100% relative humidity (RH) (Figure 1A). 
	CAUTION: Please consult with the institution&#39;s Environmental Health and Safety guidelines for the safe location of the manual plunger and recommended operations.</li>
	<li>Prior to grid preparation, turn on the humidifier in the cold room to ensure the RH of the cold room is &#8805; 95 %. 
	NOTE: Grid preparation in low humidity can result in dehydration of thin films, alteration of buffer components due to evaporation, and a decrease in grid-to-grid reproducibility46. It is not recommended to freeze grids at &#60;80 % RH.</li>
	<li>Ensure the temperature of the cold room is at 4 &#176;C.</li>
	<li>Locate the manual plunger away from strong air currents (i.e., away from air conditioning unit vents) as they can lead to turbulence near grids and/or prominent ice accumulation on cold surfaces.</li>
</ol>
2. Prepare plunging materials and accessories
NOTE: Estimated operating time: 1-5 minutes
<ol>
	<li>Use clean scissors to cut blotting paper circles into 1-1.5 cm wide and ~9 cm long strips. Avoid touching the center of the blotting paper and discard the smaller end pieces. It is important to ensure that the blotting paper strips are dry, clean, and free of contaminants. Separate the strips and place them in a 100 mm Petri dish.</li>
	<li>Place a 22x22 mm square glass coverslip into a separate 60 mm glass Petri dish. This coverslip-containing glass Petri dish will be used to store, transfer, and glow-discharge grids. 
	NOTE: It is recommended to use an air-duster can to remove any visible debris from the slide prior to adding grids. An anti-static gun can also be used to remove any static electricity that accumulates.</li>
	<li>Assemble and label grid storage boxes.</li>
	<li>Acquire 4 to 6 clean and dry clamping tweezers and locate them to the manual plunger. Visually inspect each tweezer prior to plunging to ensure they are not damaged and are free of contaminants.</li>
</ol>
3. Prepare the cryogen dewar and manual plunger
NOTE: Estimated operating time: 5-15 minutes
<ol>
	<li>Install the platform base at the bottom of the plunging dewar. Place the ethane vessel dewar on top of the platform base, add the brass ethane vessel, and then install the spinning grid storage platform.
	<ol>
		<li>Once liquid nitrogen (LN2) is added to the plunging dewar, the height of the platform base can no longer be adjusted. Ensure that the grid base is installed properly and is level to limit grid and/or tweezer damage due to improper plunger height.</li>
	</ol>
	</li>
	<li>Prior to freezing, check the manual plunger and all ancillary equipment to ensure they are functioning properly to limit sample and/or grid loss.</li>
	<li>Prior to each freezing session, replace the tape on the manual plunger arm used to hold the tweezers. The high humidity of the room can deteriorate the tape adhesive and decrease the ability of the tape to hold the tweezers, increasing the likelihood of tweezer damage and/or grid loss.</li>
	<li>Adjust the lamp(s) near the manual plunger to ensure there is sufficient light to monitor sample wicking and ensure grid transfer is easily visualized to prevent grid damage and/or loss (see step 3.7). Use a ring lamp directly behind the plunger to visualize liquid movement and a flexible arm task light near the dewar to illuminate the frozen grids.</li>
	<li>Adjust the foot pedal tension to ensure the dewar plunging arm is securely retained in place while in the raised position and is fully released when the pedal is depressed. Perform several &#34;dry&#34; runs prior to sample application to ensure the plunger is working properly. 
	NOTE: Improper adjustment of the foot pedal tension will result in premature release of the plunging arm (i.e., tension is set too low) and grid loss or incomplete plunging of the grid into the ethane vessel (i.e., the tension is set too high).</li>
	<li>Place the plunging dewar at the base of the manual plunger directly below the plunging arm and secure it in place. Attach a pair of tweezers to the plunging arm using the attached tape. While holding the manual plunging arm, depress the foot pedal to carefully lower the plunging arm and adjust the travel of the plunging arm to ensure the grid will locate within the middle of the ethane vessel.</li>
	<li>Use the bump stop at the top of the plunging arm to determine the final position of the plunging arm when the foot pedal is fully depressed (Figure 1B). Adjust the bump stop height on the plunging arm to adjust the location of the grid in the ethane vessel (Figure 1C). 
	NOTE: Improperly setting the plunging arm height can lead to the grid and/or tweezer damage (e.g., plunging arm height is set too low) or inadequate vitrification (e.g., plunging arm height is set too high).</li>
	<li>Locate the dewar outside the cold room and proceed to preparing liquid ethane (step 4).</li>
</ol>
4. Prepare the cryogen
NOTE: Estimated operating time: 10-30 minutes
<ol>
	<li>Assess the ethane tank, regulator, tubing, and ethane dispensing tip for any signs of damage. Immediately report and rectify any signs of damage before proceeding. 
	CAUTION: Compressed ethane and ethane:propane gas mixes are flammable and can pose a serious threat to life and/or result in injury if improperly handled. Please consult an expert if unsure how to operate or handle compressed gas tanks. Please refer to the institution&#39;s Environmental Health and Safety guidelines when handling flammable compressed gases. In addition, liquified ethane is a powerful cryogen that can pose a serious threat to life and/or injury if not handled properly. Please refer to the institution&#39;s Environmental Health and Safety guidelines when handling cryogens.</li>
	<li>Acquire sufficient LN2 in an appropriate LN2 handling dewar (i.e., 3-4 L is typical for grid preparation and storage). 
	CAUTION: LN2 is a cryogen that can pose a serious threat to life and/or injury if not handled properly.
	<ol>
		<li>Ensure all personal protective equipment are utilized to minimize the risk of injury. The vapor of LN2 is an asphyxiant and should be handled in well-ventilated areas. Please consult an expert if unsure how to operate or handle cryogenic vessels and cryogens. Please refer to the institution&#39;s Environmental Health and Safety guidelines when handling cryogens. For situations in which liquid nitrogen cannot be used in a cold room, we recommend plunge freezing in a cool and well-ventilated space.</li>
	</ol>
	</li>
	<li>Outside of the cold room, cool down the plunging dewar by pouring LN2 directly into the brass ethane vessel until the liquid nitrogen level reaches the top of the ethane vessel (i.e., just above the platform). Top off the LN2 outside the ethane vessel as needed. Proceed to the next step when the LN2 stops bubbling violently (approximately 5 minutes).
	<ol>
		<li>Add LN2 directly to the brass ethane vessel to sufficiently cool the vessel prior to condensing ethane gas. Failure to properly cool the brass ethane vessel will dramatically increase the time it takes to condense ethane.</li>
		<li>Once the dewar has reached LN2 temperature, avoid overfilling the dewar such that LN2 spills into the ethane vessel.</li>
	</ol>
	</li>
	<li>Ethane comes as a compressed gas and needs to be liquefied for use. The ethane tank utilizes a high-purity dual-stage regulator to control the gas flow. Connect tubing to the regulator outlet valve and use a 14-gauge flat metal dispensing tip connected at the end for dispensing ethane.</li>
	<li>Prior to opening the ethane main tank valve, make sure the pressure adjusting knob and outlet valve are closed all the way. Fully open the main tank valve and then open the outlet valve to ~50%. Slowly open the pressure adjusting knob until a slow gas flow is observed. Use the outlet valve to fine-tune the gas flow. 
	CAUTION: Always point the metal dispensing tip away from self when opening valves or making gas flow adjustments.</li>
	<li>Slowly start the gas flow and assess the flow rate by inserting the tip of the ethane gas line into a small beaker of deionized water. Adjust the gas flow until the flow rate moderately disturbs the water.
	<ol>
		<li>Adjust the gas flow rate to ensure proper ethane condensing occurs - too slow of a flow rate will cause the ethane gas to solidify in the dispensing tip and too fast of a flow rate will result in intense bubbling and prevent freezing.</li>
	</ol>
	</li>
	<li>Prior to inserting the tip of the ethane gas line into the brass ethane vessel, clean and wipe the dispensing tip with delicate task wipes to remove any water.</li>
	<li>In a smooth and quick motion, locate the gas dispensing tip at the bottom of the brass ethane vessel and begin moving the dispensing tip in a slow circle around the bottom of the ethane vessel. Solid ethane will form immediately but will quickly liquefy as more ethane gas is added/condensed.
	<ol>
		<li>Continue to move the metal ethane dispensing tip around at the bottom of the ethane vessel to liquefy the solid ethane. Fill up the ethane vessel to &#190; full of liquid ethane (2-3 threads from the top). Stop ethane gas flow by carefully removing the metal ethane dispensing tip from the brass ethane vessel and closing the outlet valve.</li>
	</ol>
	</li>
	<li>Top off the plunging dewar with LN2&#160;pouring gently on the side of the dewar to avoid LN2 addition to the brass ethane vessel, until the liquid level just touches the brass ethane vessel. Place the foam lid on the plunging dewar to facilitate ethane solidification.
	<ol>
		<li>LN2 must directly contact the brass ethane vessel to aid in the solidification of ethane. After approximately 5 minutes, the ethane within the brass vessel will become completely frozen solid. Add more LN2 until it just touches the ethane vessel and proceed to the next step.</li>
	</ol>
	</li>
	<li>If the ethane has not frozen solid, then the brass ethane vessel is not cold enough for the remaining steps. Add more LN2 until it just touches the ethane vessel and wait an additional 5 minutes. Ensure that the ethane within the brass vessel is completely frozen solid.</li>
	<li>Open the gas outlet valve to produce an ethane gas flow at a similar rate as determined in step 4.6. Vertically place the metal ethane dispensing tip into the solid ethane and continue to move the ethane dispensing tip in a circular motion to melt the solid ethane.
	<ol>
		<li>Continue to add ethane until it is level with the top of the brass ethane vessel. Slowly remove the tip from the ethane vessel and close the ethane tank outlet valve. Cover the dewar with the lid for ~1-4 minute(s) to let the ethane solidify around the edges of the brass ethane vessel. 
		NOTE: An ideal ethane vessel will have a 2-3 mm symmetric ring of solid ethane at the perimeter of the brass ethane vessel with liquid ethane in the center (Figure 1D and Figure 2).</li>
		<li>Ensure that the ethane be as cold as possible without solidifying to ensure proper vitrification of the biological specimen. Failure to properly prepare the liquid ethane can result in inadequate vitrification of the specimen, ice accumulation, and/or loss of specimen, each contributing to the deterioration of specimen quality for imaging.</li>
		<li>If a solid ring of ethane does not form after 2-3 minutes, add more LN2 to the dewar and cover for 2-5 more minutes.</li>
		<li>If the ethane is solidifying too quickly, then use a large pair of clean tweezers to gently warm the ethane vessel and/or solid ethane to prevent complete ethane solidification. Once the ethane and LN2 are stable, close all the ethane tank valves and locate the plunging dewar to the manual plunger. Secure the plunging dewar to the manual plunger. 
		CAUTION: Be extra careful when transferring the dewar as LN2 can spill into the ethane vessel and solidify the liquid ethane. If need be, a clean set of tweezers can be used to melt any solid ethane in the middle of the vessel.</li>
	</ol>
	</li>
	<li>Test the location of the ethane dewar with a pair of empty tweezers to ensure the tweezer tip locates at the center of the ethane vessel and there is sufficient space for the grid and tweezer tip inside the liquid ethane (Figure 1D).
	<ol>
		<li>If the solid ethane ring is too thick for easy grid handling, then use a pair of room temperature, clean tweezers to melt the solid ethane and create more freezing area at the center of the ethane vessel.</li>
	</ol>
	</li>
</ol>
5. Prepare EM grids
NOTE: Estimated operating time: 1-5 minutes
<ol>
	<li>Add the grid storage box(es) to the dewar, unscrew the grid storage box lid(s), and make sure each lid can freely rotate to a new grid slot.</li>
	<li>Carefully transfer grids from the grid storage box to the edge of the square glass coverslip with ~30-40% of the grid off the slide edge. Ensure that the grid foil is facing up. Ensure grids are covered when not in use. Placing the grids over the edge of the square glass coverslip provides ease of grid handling and decreases the chance of bending or damaging the grid during transfer. 
	NOTE: 4-6 grids are typically prepared at a time.</li>
	<li>Render grids hydrophilic using a glow discharger or plasma cleaner. 
	NOTE: Please refer to the recommended guidelines for grid cleaning provided by the glow discharger/plasma cleaner manufacturer.
	<ol>
		<li>Use the grids within 10 minutes of plasma cleaning as the grids lose hydrophilicity and grid-to-grid reproducibility decreases after this time.</li>
	</ol>
	</li>
</ol>
6. Prepare cryoEM specimen by plunge freezing
NOTE: Estimated operating time: &#62;10 minutes (~1-3 min per grid)
<ol>
	<li>Use a clean and dry clamping tweezer to pick up a cleaned grid, slide the plastic clamp down to fix the grid in place, and gently tap the tweezers to ensure the grid is properly secured.
	<ol>
		<li>Handle grids by the outer ring to prevent damage to the grid foil.</li>
	</ol>
	</li>
	<li>Apply 1 to 5 &#181;L of sample to the prepared side (e.g., front or foil side) of the grid. 
	NOTE: The optimal volume and blotting time depends on the sample and needs to be optimized for each sample; larger volumes and more viscous samples require longer blotting times.</li>
	<li>Secure the tweezer-grid-sample assembly to the manual plunging arm by wrapping tape around the tweezer handle.
	<ol>
		<li>Face the sample towards the user for traditional front blotting. If the sample requires back blotting, then locate the sample away from the user.</li>
	</ol>
	</li>
	<li>Hold a clean, dry, cut piece of blotting paper between the thumb and index finger of each hand.
	<ol>
		<li>Only handle the blotting papers from the edges and never touch the center as oils and other contaminants from hands/gloves can alter grid quality.</li>
	</ol>
	</li>
	<li>Rest hands on the edge of the plunging dewar to establish a stable position. Locate the blotting paper parallel to the grid surface approximately 1 cm away from the grid surface.
	<ol>
		<li>Use the middle section of the blotting paper to allow for complete fluid mobility and even wicking across the grid surface.</li>
	</ol>
	</li>
	<li>Gently slide and rotate the thumb and ring fingers towards each other to bend blotting paper toward the grid to initiate blotting. Maintain contact between the blotting paper and the grid surface during the entire blotting process.
	<ol>
		<li>Directly contact the blotting paper to the grid surface and maintain consistent contact across the grid surface.</li>
		<li>Gently bending the blotting paper decreases bending of the grid and/or damage to the grid surface, and results in more consistent ice across the grid (Figure 3A).</li>
	</ol>
	</li>
	<li>Observe the mobile liquid front and once it stops progressing into the blotting paper begin counting for 4 to 6 seconds. 
	NOTE: Counting can occur once the blotting paper contacts the grid surface but lower grid-to-grid reproducibility can occur. The total blot time will depend on the grid type, foil type, sample concentration, and sample type (e.g., soluble versus membrane versus filamentous proteins). For more viscous samples longer blot times (e.g.., 5 to 7 seconds) will be required.
	<ol>
		<li>Important: Develop a reliable and consistent counting scheme to greatly enhance reproducibility during the freezing process.</li>
	</ol>
	</li>
	<li>Move the left, right thumb and index fingers in opposite directions to remove the blotting paper in a &#34;snapping motion&#34; away from the grid surface. Immediately depress the foot pedal to release the plunging arm and plunge the grid into liquid ethane.
	<ol>
		<li>Simultaneously remove the blotting paper and press the foot pedal to plunge the grid into the ethane as soon as possible for best freezing results. The longer the time between blotting paper removal and plunging, the more evaporation of thin films will occur and decrease grid-to-grid reproducibility.</li>
	</ol>
	</li>
	<li>Use one hand to stabilize the clamping tweezers, unwind tape carefully from around the tweezers and manual plunging arm.
	<ol>
		<li>Always maintain contact with the tweezer to prevent movement of the tweezer-grid and limit grid damage that occur from knocking the grid against the solid ethane.</li>
	</ol>
	</li>
	<li>Once clamping tweezers are free from the manual plunging arm, maintain the tweezer in one hand resting on top of the plunging dewar, ensure the grid remains in the liquid ethane. Carefully slide the plastic clamp away from the grid so the grid can be transferred. Maintain pressure on the tweezers to retain the grid.
	<ol>
		<li>With one swift motion, quickly transfer the grid from the ethane vessel into the LN2 reservoir. Carefully place the grid in the grid storage box. 
		NOTE: Some ethane may solidify on the grid surface. Opening the tweezer slightly will break the ethane and allow for the grid to be dropped into the grid box.</li>
	</ol>
	</li>
	<li>Wrap the tip of the tweezers in a delicate task wipe to prevent frost accumulation. Set aside until the tweezers have returned to room temperature.
	<ol>
		<li>Have 4 to 6 tweezers on hand for ease of use. Each tweezer will be used for sample freezing and warmed before subsequent use.</li>
	</ol>
	</li>
	<li>Repeat steps 6.1-6.11 for each grid.</li>
	<li>Once freezing has culminated, securely close the grid box and transfer to a proper storage location.</li>
	<li>Carefully dispose of the liquid ethane and LN2 and store all materials in a dry location.</li>
</ol>

We have nothing to disclose.

The vitrification of biological specimens for imaging by single-particle cryogenic electron microscopy (cryoEM) remains a critically important step for successful structure determination. The manual blot-and-plunge method described in this protocol represents a cost-effective, reliable, and robust method for quickly freezing biological samples in thin films of vitreous ice for cryoEM imaging. Using the methods outlined in the manuscript, researchers will be able to assemble and operate the manual plunger, prepare cryogen...

Single-particle cryogenic electron microscopy (cryoEM) is a powerful structural technique that can be used to solve structures of dynamic biological specimens to near-atomic resolution1,2,3,4. Indeed, recent advances in direct electron detector technologies4,5,6,7,8,9,10, improvements in electron sources4,11,12,13,14, and electromagnetic lens stability15, coupled with the continued development of data acquisition16,17 and analysis software packages18,19, have enabled researchers to now routinely determine structures of well-behaved specimens to 3 &#197; resolution or better4,11,13,14,20,21,22,23. Despite these improved imaging and data processing capabilities, cryoEM grid preparation remains the largest impediment for successful high-resolution structure determination and often serves as a considerable bottleneck in the EM workflow24,25,26,27.
CryoEM relies on the imaging of biological samples in aqueous solutions that are frozen to form a thin film of &#34;glass-like&#34; ice - a process known as vitrification - that preserves the native biochemical state. Vitrification of biological samples for cryoEM dates back over 40 years28,29,30 and many techniques and equipment that have been developed for this process rely on the originally detailed blot-and-plunge method31,32,33,34,35, whereby a small volume of sample (e.g., 1-5 &#181;L) is applied to a specialized EM grid before the excess solution is removed using physical interaction of the grid with blotting paper. The timing of this process is usually empirically determined for each specimen as a critical component of freezing samples is the thickness of the vitreous ice film - if the ice is too thick then imaging quality deteriorates dramatically due to increased scattering of the electron beam while ice that is too thin can restrict protein orientations and/or exclude particles from the center of the grid foil holes36. This reliance on the perfect ice thickness for single-particle cryoEM has led to a wide array of techniques and equipment that can freeze samples, including robotics37,38, microfluidics42, and ultrasonic or spraying devices27,39,40,41,42,43,44. In recent years, some of the most popular sample preparation devices rely on the use of robotics for automated freezing of samples using the blot-and-plunge technique45. While these devices are designed to reproducibly create proper ice thickness for imaging, they often remain too expensive for individual labs to purchase and operate and are generally found within cryoEM facilities at hourly rates for usage. In recent years, the original manual blot-and-plunge technique has come back into increased use3,47,48,49,50,51,52. Indeed, a manually operated blot-and-plunge device can achieve high-quality cryoEM grids at a fraction of the cost of robotic counterparts.&#160;Furthermore, manual blotting also offers more users control over blotting as researchers can adjust the type of blotting (i.e., back-blotting of the grid, front-blotting of the grid, etc.), and blotting time based on each individual sample and research questions.
In this article, we provide details on how to effectively freeze biological samples using a traditional manual blot-and-plunge vitrification device coupled with a custom-designed dewar platform53. Best practices, including preparation of the cryogen, grid handling, sample application, and blotting, as well as common pitfalls and recommendations on how to overcome these hurdles are provided. Advice on how to increase ice thickness reproducibility between grid preparations and how to modify sample blotting based on biological specimen type are discussed. Given the low cost associated with the purchase and operation of the manual plunger described in this manuscript, labs across the globe can prepare biological specimens for cryoEM in a cost-effective and reproducible manner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4 slot grid storage box</td>
			<td>Ted Pella</td>
			<td>160-40</td>
			<td></td>
		</tr>
		<tr>
			<td>14 gauge flat metal dispensing tip</td>
			<td>Amazon</td>
			<td>B07M7YWWLT</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm square glass coverslip</td>
			<td>Sigma</td>
			<td>C9802-1PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm glass Petri dish to store grids</td>
			<td>Fisher</td>
			<td>08-747A</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm glass Petri dish to store Whatman paper</td>
			<td>Fisher</td>
			<td>08-747D</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm glass Petri dish to store Whatman paper</td>
			<td>Fisher</td>
			<td>08-747F</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL beaker</td>
			<td>Fisher</td>
			<td>02-555-25B</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue styrofoam dewar</td>
			<td>Spear Lab</td>
			<td>FD-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass ethane vessel</td>
			<td>Lasco</td>
			<td>17-4075</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping tweezers</td>
			<td>Ted Pella</td>
			<td>38825</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipes</td>
			<td>Fisher</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-stage regulator with control valve</td>
			<td>Airgas</td>
			<td>Y12N245D580-AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dewer grid base</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane platform</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethane propane tank</td>
			<td>Praxair</td>
			<td>ET PR50ZU-G</td>
			<td>ethane (50%) : propane (50%) in a high-pressure tank</td>
		</tr>
		<tr>
			<td>Ethane tank</td>
			<td>Praxair</td>
			<td>UN1035</td>
			<td>ethane (100%)</td>
		</tr>
		<tr>
			<td>Flexible arm task light</td>
			<td>Amscope</td>
			<td>LED-11CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Grids (UltrAufoil R 1.2/1.3 300 mesh)</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q325AR1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidifier</td>
			<td>Target</td>
			<td>719438</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygrometer</td>
			<td>ThermoPro</td>
			<td>B01H1R0K68</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab coat</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen dewar</td>
			<td>Worthington</td>
			<td>LD4</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen gloves</td>
			<td>Fisher</td>
			<td>19-059-925</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual plunger stand (black stand + foot pedal)</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mark 5 (plunging platform)</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>VWR</td>
			<td>82026-424</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipette</td>
			<td>Eppendorf</td>
			<td>13-690-029</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Eppendorf</td>
			<td>E0030124286</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>ibis scientific</td>
			<td>63300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring lamp</td>
			<td>Amazon</td>
			<td>B07HMR4H8G</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety glasses</td>
			<td>UCSD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Amazon</td>
			<td>Fiskars 01-004761J</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw driver</td>
			<td>Ironside</td>
			<td>354711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Fisher</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer to transfer grid box</td>
			<td>Amazon</td>
			<td>LTS-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher</td>
			<td>14-171-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman blotting paper</td>
			<td>Fisher</td>
			<td>1001-090</td>
			<td></td>
		</tr>
	</tbody>
</table>

manual blot-and-plunge freezing of biological specimens for single-particle cryogenic electron microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

Successful execution of the blot-and-plunge protocol described here will result in a thin, uniform layer of vitreous ice that is free of any hexagonal ice, contaminants, and large gradients of unusable ice which can be observed under the electron microscope (Figure 3). Inconsistent contact of the blotting paper with the grid surface, prematurely removing the blotting paper, or moving the blotting paper during grid contact can decrease the quality of the vitreous ice and lead to inconsistent ...

Watch this Scientific Journal Video about Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy at JoVE.com

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

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Research

JoVE Journal

Biochemistry

6.0K Views.  University of California, San Diego. This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Video: Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Analysis of &amp;#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

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 ...

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.

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 ...

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 ...

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 ...

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 ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

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 ...