The authors thank Dr. Herv&#233; Remigy of Thermo Fisher Scientific for useful advice. The cryo-EM experiments were performed at the Academia Sinica Cryo-EM Facility (ASCEM). ASCEM is supported by Academia Sinica (Grant No. AS-CFII-108-110) and Taiwan Protein Project (Grant No. AS-KPQ-109-TPP2). The authors also thank Ms. Hui-Ju Huang for the assistance with the sample preparation.

NOTE: This protocol aims to use a modified commercial vitrification apparatus to prepare the cryo-electron microscopy (cryo-EM) samples at specific temperatures, especially higher than 37 &#176;C. The overall experimental setup is shown in Figure 1. The protocol uses 55 &#176;C as an example. For the specific conditions at other temperatures, please refer to Supplementary Table 2 in reference12.
1. Preparation of the vitrification apparatus
<ol>
	<li>Make a 1 cm hole in a 50 mL centrifuge tube on its closed end.</li>
	<li>Place the tube in the vitrification apparatus chamber at the ultrasonic water outlet, as shown in Figure 2. 
	NOTE: The purpose is to minimize water condensation by guiding water vapor to the heat exchanger through the tube before reaching the entire chamber.</li>
	<li>Set up the vitrification apparatus temperature to the specified temperature (e.g., 55 &#176;C, as shown in Figure 3), and allow the vitrification apparatus chamber to reach to 55 &#176;C and 100% relative humidity. Let it stand for at least half an hour to stabilize the conditions before starting the experiment.</li>
</ol>
2. Warming up the sample and the tools
<ol>
	<li>Place the water bath on a hotplate and set the hotplate to the desired temperature (here 55 &#176;C). Check with a thermometer to ensure that the water reaches 55 &#176;C.</li>
	<li>Incubate the sample in the water bath and preheat the pipette tip on the edge of the hotplate for 2 min or longer before the blotting experiment. 
	&#8203;NOTE: The highest temperature setting for the vitrification apparatus chamber is 60 &#176;C. To prepare higher temperature grids for cyro-EM experiment (e.g., 70 &#176;C), the sample is incubated in the water bath at 80 &#176;C, and the average between the sample temperature and the vitrification apparatus temperature is estimated to be the actual temperature of the sample on the grid (70 &#176;C in this case). See the Discussion section for further details and limitations on this estimation.</li>
</ol>
3. Preparation for the blotting experiment
<ol>
	<li>Glow discharge a holey carbon-supported grid at 25 mA for 30 s, or alternative to the values depending on the device used.</li>
	<li>Incubate the tweezers with the grid in the vitrification apparatus for 2 min or longer.</li>
	<li>Fill the ethane container with ethane according to standard procedures. Do not let the ethane overflow. 
	NOTE: This step takes about 10 min and should be followed immediately with the blotting experiment to avoid freezing.</li>
	<li>Place the vitrification filter paper into the vitrification apparatus chamber no sooner than 5 min before the blotting experiment. 
	NOTE: Placing the filter paper in the chamber too soon will cause it to become too wet.</li>
</ol>
4. Blotting experiment
NOTE: When holding the grid, ensure that the grid is stable and there is a minimal contact area with the tweezers (Figure 4). This is done to maintain the best cooling efficiency of ethane and to avoid non-vitreous ice.
<ol>
	<li>Use a pipette tip to apply 7-9 &#956;L of the sample to the grid. Then wait for 1-2 s, blot for 1-1.5 s, and quickly plunge the sample to liquid ethane.</li>
	<li>Transfer the grid from liquid ethane to the cryo box, which is stored in liquid nitrogen. 
	&#8203;NOTE: This step must be performed very carefully because the tweezers is still hot, so the entire cooling tank is full of vapor at this time.</li>
</ol>
5. Quality check for the grids
<ol>
	<li>Clip the grids and upload them to a cryo-EM instrument.</li>
	<li>Use the display screen of the cryo-EM instrument and the low dose function of the software to screen the ice condition on the grid and the distribution of the sample on the grid. 
	&#8203;NOTE: Often, the grids are very dry, or the ice layer is too thick. The success rate of the grids prepared at high temperatures is substantially lower than that at room temperature or 4 &#176;C. Representative results of good grids and non-satisfactory grids are shown in the next section.</li>
	<li>If the quality of the resulting grid is not good, repeat the process of grid preparation with varied conditions (such as waiting time, blotting time, etc.). If the quality of the resulting grid is good, repeat the same steps to make grids at a different temperature.</li>
</ol>
6. Data collection
<ol>
	<li>Transfer the good quality grids to a high resolution cryo-EM instrument.</li>
	<li>Perform data collection and data analyses according to established procedures. 
	NOTE: As shown in our previous publication, the resolution of the structure is not affected by the high temperature12.</li>
</ol>

<ol><li>Yip, K. M., Fischer, N., Paknia, E., Chari, A., Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic-resolution+protein+structure+determination+by+cryo-EM%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Atomic-resolution protein structure determination by cryo-EM.</a> Nature. 587, 157-161 (2020).</li>
<li>Nakane, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-particle+cryo-EM+at+atomic+resolution%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-particle cryo-EM at atomic resolution.</a> Nature. 587, 152-156 (2020).</li>
<li>Chen, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Cryo-EM+to+uncover+structural+bases+of+pH+effect+and+cofactor+bi-specificity+of+ketol-acid+reductoisomerase%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Use of Cryo-EM to uncover structural bases of pH effect and cofactor bi-specificity of ketol-acid reductoisomerase.</a> Journal of the American Chemical Society. 141, 6136-6140 (2019).</li>
<li>Cabra, V., Samsó, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do%27s+and+don%27ts+of+cryo-electron+microscopy%3A+A+primer+on+sample+preparation+and+high+quality+data+collection+for+macromolecular+3D+reconstruction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Do's and don'ts of cryo-electron microscopy: A primer on sample preparation and high quality data collection for macromolecular 3D reconstruction.</a> Journal of Visualized Experiments. (95), e52311(2015).</li>
<li>Klebl, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Need+for+speed%3A+Examining+protein+behavior+during+CryoEM+grid+preparation+at+different+timescales%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Need for speed: Examining protein behavior during CryoEM grid preparation at different timescales.</a> Structure. 28 (11), 1238-1248 (2020).</li>
<li>Passmore, L. A., Russo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specimen+preparation+for+high+resolution+cryo-EM%5BTitle%5D)+AND+%22J.+Methods+in+Enzymology%22%5BJournal%5D)">Specimen preparation for high resolution cryo-EM.</a> J. Methods in Enzymology. 579, 51-86 (2016).</li>
<li>Laughlin, T. G., Bayne, A. N., Trempe, J. -F., Savage, D. F., Davies, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+complex+I-like+molecule+NDH+of+oxygenic+photosynthesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the complex I-like molecule NDH of oxygenic photosynthesis.</a> Nature. 566, 411-414 (2019).</li>
<li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures+and+operating+principles+of+the+replisome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Structures and operating principles of the replisome.</a> Science. 363, (2019).</li>
<li>Zhao, Y., Chen, S., Swensen, A. C., Qian, W. -J., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Architecture+and+subunit+arrangement+of+native+AMPA+receptors+elucidated+by+cryo-EM%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM.</a> Science. 364, 355-362 (2019).</li>
<li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+dynamics+of+ribosome+subunit+association+studied+by+mixing-spraying+time-resolved+cryogenic+electron+microscopy%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.</a> Structure. 23, 1097-1105 (2015).</li>
<li>Singh, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+of+temperature+sensation+by+the+TRP+channel+TRPV3%5BTitle%5D)+AND+%22Nature+Structure+and+Molecular+Biology%22%5BJournal%5D)">Structural basis of temperature sensation by the TRP channel TRPV3.</a> Nature Structure and Molecular Biology. 26, 994-998 (2019).</li>
<li>Chen, C. Y., Chang, Y. C., Lin, B. L., Huang, C. H., Tsai, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-resolved+cryo-EM+uncovers+structural+bases+of+temperature-dependent+enzyme+functions%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Temperature-resolved cryo-EM uncovers structural bases of temperature-dependent enzyme functions.</a> Journal of the American Chemical Society. 141, 19983-19987 (2019).</li>
</ol>

The authors declare no competing financial interests.

In step 1 of the protocol, make sure that the centrifuge tube has been installed well and does not fall when the experiment is in progress. Due to the accumulation of a large number of water droplets in the chamber, which could change the adsorption capacity of the filter paper, it is recommended that the overall time of the experiment should not exceed 30 min after the vitrification apparatus chamber reached the equilibration temperature. If the operation time exceeds 30 min, the operator needs to replace the filter pap...

The cryo-EM technology for solving the structures of protein complexes has continued to improve, particularly in the direction of obtaining high-resolution structures1,2. In the meantime, the landscape of its application has also been expanded by varying the sample conditions such as pH or ligands prior to the vitrification process3, which involves the preparation of sample grids followed by plunge freezing4,5. Another important condition is the temperature. Although cryo-EM experiments, like X-ray crystallography, are performed at low temperatures, the structure solved by cryo-EM reflects the structure at the solution state prior to vitrification. Until recently, the majority of single particle analysis (SPA) cryo-EM studies use samples that are kept on ice (i.e., at 4 &#176;C) prior to vitrification6, though a number of studies use samples at around room temperature7,8,9,10 or as high as 42 &#176;C11. In a recent report, we performed temperature-dependent studies of the enzyme ketol-acid reductoisomerase (KARI) from the thermophilic archaeon Sulfolobus solfataricus (Sso) at six different temperatures from 4 &#176;C to 70&#176;C12. Our studies suggest that it is important to prepare sample grids at functionally relevant temperatures and that cryo-EM is the only structural method that is practically feasible for solving the structure of the same protein complex at multiple temperatures.
The major difficulty for vitrification at high temperatures is to minimize vapor condensation and achieve thin ice. Here we report the detailed protocol used for preparing sample grids at high temperatures in our previous study of the Sso-KARI12. We assume that the readers or viewers are already experienced in the overall sample preparation and data processing procedures for cryo-EM experiments and emphasize the aspects relevant to high temperature.

<table><tbody><tr><td>Falcon tube</td><td>Falcon</td><td>352070</td><td>size: 50 mL</td></tr><tr><td>Filter paper</td><td>Ted Pella</td><td>47000-100</td><td>&amp;Oslash;55/20mm, Grade 595</td></tr><tr><td>HI1210</td><td>Leica</td><td></td><td>water bath</td></tr><tr><td>K100X</td><td>Electron Microscopy Sciences</td><td></td><td>glow discharge</td></tr><tr><td>Quantifoil, 1.2/1.3 200Mesh Cu grid</td><td>Ted Pella</td><td>658-200-CU-100</td><td></td></tr><tr><td>Titan Krios G3</td><td>Thermo Fisher Scientific</td><td>1063996</td><td>low dose imaging</td></tr><tr><td>Vitrobot Mark IV</td><td>Thermo Fisher Scientific</td><td>1086439</td><td></td></tr><tr><td>Vitrobot Tweezer</td><td>Ted Pella</td><td>47000-500</td><td></td></tr></tbody></table>

preparation of high-temperature sample grids for cryo-em

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

The low magnification overview is shown in Figure 5A,B. Panel A is an example of a successful grid. There is an ice gradient from top left (thicker) to bottom right (thinner or empty). Such a grid makes it easier to find an appropriate thickness of the ice layer in the middle area suitable for data collection, such as the blue and green boxes. The grid B is too dry. The squares in the grid have bright contrast, which means that the ice layer is too thin or there is no ice la...

Watch this Scientific Journal Video about Preparation of High-Temperature Sample Grids for Cryo-EM at JoVE.com

Preparation of High-Temperature Sample Grids for Cryo-EM

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

preparation-of-high-temperature-sample-grids-for-cryo-em

Research

JoVE Journal

Biochemistry

3.5K Views.  Academia Sinica. De functies van eiwitten zijn temperatuurafhankelijk en zijn optimaal bij de temperatuur. Dit protocol biedt een manier om eiwitstructuren bij hogere temperaturen op te lossen. Deze aanpak kan functioneel relevante structuren bieden en het begrip van de temperatuurafhankelijkheid van eiwitstructuren vergroten.De procedure wordt gedemonstreerd door Yuan-Chih Chang, manager van de Academia Sinica Cryo EM-faciliteit. Begin met het maken van een gat van één centimeter in een centrifugebu...

Video: Preparation of High-Temperature Sample Grids for Cryo-EM

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 ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

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 ...

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.

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 ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biology

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

Coating TEM Grids with a Graphene Monolayer

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Author Spotlight: Enhancing CryoEM Resolution Using Graphene-Coated Grids 

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.

Preparation of High-Temperature Sample Grids for Cryo-EM

Samenvatting

Meer video's verkennen