Cryo-EM sample preparation

High-quality protein preparation is the foundation of any successful structural biology technique. For cryo-electron microscopy (cryo-EM), biophysical aspects such as composition, purity, homogeneity, and stability, as well as biochemical activity strongly contribute to preparation of good quality cryo-EM grids, but they can also significantly impact the resolution of the subsequent computational reconstruction. Starting with protein expression, followed by solubilization and stabilization of the expressed protein, with subsequent purification and clean-up, Thermo Fisher Scientific offers a wide range of solutions to achieve the highest sample quality before freezing of the specimen on the EM grid.


Follow these 5 steps for successful cryo-EM sample preparation

Get started in single-particle analysis

Want to learn how to prepare your samples for cryo-EM or how to operate an electron microscope? Visit the EM–learning platform that features over 70 hours of theoretical lectures and videos.

Are you interested in hands-on experience with cryo-EM sample preparation? Try the Thermo Scientific VitroEase Cryo-EM Training Kit. This vitrification starter kit includes everything you need to immediately start preparing your first couple of grids, including a detailed walkthrough manual. 

Download our Scientific Workflows app for Android or iOS for a step-by-step visual workflow guide.

Protein preparation for structural biology

Any high-resolution cryo-EM structure starts with good protein preparation. Check out our Protein Sample Preparation eLearning Course to find out more about high-quality preparation of cryo-EM protein samples. 

Interested in further optimizing your protein production, purification, or biochemistry? Our sample preparation methods page contains a wide range of protein analysis techniques organized by sample type and method.  

After your sample is purified, it is important to assess whether the purification quality is suitable for further electron-microscopy analysis. Standard biochemical methods are not entirely sufficient, as solutions of intact complexes can actually consist of mixtures of compositionally different sub-complexes or structurally different conformations.

Negative-stain transmission electron microscopy

Negative-stain electron microscopy is an easy and straightforward method to assess the quality of purified biological specimens at the microscopic scale. The objective of this screening is to qualitatively assess particle composition and conformational homogeneity, which can only be done at the microscopic scale. Prepare negative stained samples more easily with ready-to-use VitroEase Methylamine Vanadate Negative Stain and VitroEase Methylamine Tungstate Negative Stain

Often this assessment is done on a simple side-entry microscope (e.g., a Thermo Scientific Talos L120C TEM), since screening is usually done one grid at a time, and the actual time spent on the microscope is short.

Cryo-EM sample conditions

Cryo-EM specimens are typically prepared using several microliters of protein solution at a concentration of 50 nM to 5 μM depending on the specimen, EM grid type, and other conditions used.

Single-particle analysis with cryo-EM depends on the computational averaging of thousands of images of identical particles, and therefore structural heterogeneity should be minimized in order to simplify structure determination.

Although the single particle analysis workflow can alleviate partial heterogeneity in the specimen with 3D classification procedures, biochemical purification of the sample (to isolate the target proteins) is required.

Apart from sample heterogeneity, it is also important that the particles on the cryo-EM grid are randomly orientated, so that views from all sides are available for reconstruction. Especially with asymmetric structures, preferred orientation can be problematic and significantly hinder the resolution of your structure. In these cases, adding in low concentrations of cryo-EM compatible detergents can help overcome this problem. Furthermore, detergents can help protect the proteins during the vitrification process from the air-water interface, which might denature unstable proteins so that they are no longer in their native state.

The VitroEase Buffer Screening Kit makes optimizing your cryo-EM samples easy by providing a selection of cryo-EM-compatible buffers and detergents that can be spiked directly into your samples for minimal sample manipulation and dilution.

EM sample preparation

For compatibility with the electron microscope vacuum, and to lock the individual particles in their native states, the solution containing the sample material must be frozen. In order to preserve the macromolecular structures, freezing has to happen rapidly enough to avoid crystalline ice formation; during vitrification an amorphous solid forms instead that does little or no damage to the sample structure. Afterwards, the sample must be kept at liquid nitrogen temperatures at all times to preserve the amorphous nature of the embedding ice layer and to avoid damage to the biological particles.

Vitrification

This operation produces a frozen hydrated sample, where the individual molecules of the specimen are well distributed and embedded in a very thin layer of amorphous (vitreous) ice.

The whole procedure can be simplified using semi-automated plungers such as the Thermo Scientific Vitrobot System and can be combined with the VitroEase Cryo-EM Training Kit, which can help users learn the intricacies of the vitrification process thanks to its detailed visual manual.

Sample quality in cryo-EM

Once the sample is vitrified, it should be evaluated with diagnostic cryo-EM before high-resolution data acquisition begins. The objective of this step is to qualitatively assess if the sample is a promising target for 2D class average analysis and, simultaneously, to obtain an initial low-resolution map. During this step the sample is pre-screened to evaluate the following properties:

  • Protein concentration, stability, and distribution
  • Ice quality, thickness, and uniformity across the grid

Cryo-electron microscopy screening

This screening ideally occurs on an Autoloader-based cryo-TEM system, as multiple freezing conditions can be screened without compromising ice quality. The Thermo Scientific Glacios Cryo-TEMThermo Scientific Tundra Cryo-TEM, and Thermo Scientific Talos Arctica Cryo-TEM are best suited for this purpose.

If the sample is promising, a larger set of images may be acquired to facilitate further 2D and 3D analyses. At this stage only a moderate resolution 3D map (> 3Å) is required.

Additionally, it has been shown that a 200 kV cryo-TEM equipped with direct detector cameras can produce high-resolution (<3 Å) data.

Glacios, Tundra, and Talos Arctica Cryo-TEMs microscopes are ideally suited for this data acquisition and offer a robust and contamination-free designed-in connectivity with the higher resolution Krios Cryo-TEM. This allows for the exchange of AutoGrid cassettes and capsules between all Autoloader-equipped instruments.

Get started in single-particle analysis

Want to learn how to prepare your samples for cryo-EM or how to operate an electron microscope? Visit the EM–learning platform that features over 70 hours of theoretical lectures and videos.

Are you interested in hands-on experience with cryo-EM sample preparation? Try the Thermo Scientific VitroEase Cryo-EM Training Kit. This vitrification starter kit includes everything you need to immediately start preparing your first couple of grids, including a detailed walkthrough manual. 

Download our Scientific Workflows app for Android or iOS for a step-by-step visual workflow guide.

Protein preparation for structural biology

Any high-resolution cryo-EM structure starts with good protein preparation. Check out our Protein Sample Preparation eLearning Course to find out more about high-quality preparation of cryo-EM protein samples. 

Interested in further optimizing your protein production, purification, or biochemistry? Our sample preparation methods page contains a wide range of protein analysis techniques organized by sample type and method.  

After your sample is purified, it is important to assess whether the purification quality is suitable for further electron-microscopy analysis. Standard biochemical methods are not entirely sufficient, as solutions of intact complexes can actually consist of mixtures of compositionally different sub-complexes or structurally different conformations.

Negative-stain transmission electron microscopy

Negative-stain electron microscopy is an easy and straightforward method to assess the quality of purified biological specimens at the microscopic scale. The objective of this screening is to qualitatively assess particle composition and conformational homogeneity, which can only be done at the microscopic scale. Prepare negative stained samples more easily with ready-to-use VitroEase Methylamine Vanadate Negative Stain and VitroEase Methylamine Tungstate Negative Stain

Often this assessment is done on a simple side-entry microscope (e.g., a Thermo Scientific Talos L120C TEM), since screening is usually done one grid at a time, and the actual time spent on the microscope is short.

Cryo-EM sample conditions

Cryo-EM specimens are typically prepared using several microliters of protein solution at a concentration of 50 nM to 5 μM depending on the specimen, EM grid type, and other conditions used.

Single-particle analysis with cryo-EM depends on the computational averaging of thousands of images of identical particles, and therefore structural heterogeneity should be minimized in order to simplify structure determination.

Although the single particle analysis workflow can alleviate partial heterogeneity in the specimen with 3D classification procedures, biochemical purification of the sample (to isolate the target proteins) is required.

Apart from sample heterogeneity, it is also important that the particles on the cryo-EM grid are randomly orientated, so that views from all sides are available for reconstruction. Especially with asymmetric structures, preferred orientation can be problematic and significantly hinder the resolution of your structure. In these cases, adding in low concentrations of cryo-EM compatible detergents can help overcome this problem. Furthermore, detergents can help protect the proteins during the vitrification process from the air-water interface, which might denature unstable proteins so that they are no longer in their native state.

The VitroEase Buffer Screening Kit makes optimizing your cryo-EM samples easy by providing a selection of cryo-EM-compatible buffers and detergents that can be spiked directly into your samples for minimal sample manipulation and dilution.

EM sample preparation

For compatibility with the electron microscope vacuum, and to lock the individual particles in their native states, the solution containing the sample material must be frozen. In order to preserve the macromolecular structures, freezing has to happen rapidly enough to avoid crystalline ice formation; during vitrification an amorphous solid forms instead that does little or no damage to the sample structure. Afterwards, the sample must be kept at liquid nitrogen temperatures at all times to preserve the amorphous nature of the embedding ice layer and to avoid damage to the biological particles.

Vitrification

This operation produces a frozen hydrated sample, where the individual molecules of the specimen are well distributed and embedded in a very thin layer of amorphous (vitreous) ice.

The whole procedure can be simplified using semi-automated plungers such as the Thermo Scientific Vitrobot System and can be combined with the VitroEase Cryo-EM Training Kit, which can help users learn the intricacies of the vitrification process thanks to its detailed visual manual.

Sample quality in cryo-EM

Once the sample is vitrified, it should be evaluated with diagnostic cryo-EM before high-resolution data acquisition begins. The objective of this step is to qualitatively assess if the sample is a promising target for 2D class average analysis and, simultaneously, to obtain an initial low-resolution map. During this step the sample is pre-screened to evaluate the following properties:

  • Protein concentration, stability, and distribution
  • Ice quality, thickness, and uniformity across the grid

Cryo-electron microscopy screening

This screening ideally occurs on an Autoloader-based cryo-TEM system, as multiple freezing conditions can be screened without compromising ice quality. The Thermo Scientific Glacios Cryo-TEMThermo Scientific Tundra Cryo-TEM, and Thermo Scientific Talos Arctica Cryo-TEM are best suited for this purpose.

If the sample is promising, a larger set of images may be acquired to facilitate further 2D and 3D analyses. At this stage only a moderate resolution 3D map (> 3Å) is required.

Additionally, it has been shown that a 200 kV cryo-TEM equipped with direct detector cameras can produce high-resolution (<3 Å) data.

Glacios, Tundra, and Talos Arctica Cryo-TEMs microscopes are ideally suited for this data acquisition and offer a robust and contamination-free designed-in connectivity with the higher resolution Krios Cryo-TEM. This allows for the exchange of AutoGrid cassettes and capsules between all Autoloader-equipped instruments.


Resources

How to improve successful grid preparation through biochemistry and screening

Sample preparation and optimization for cryo-EM using the Vitrobot System

How to improve successful grid preparation through biochemistry and screening

Sample preparation and optimization for cryo-EM using the Vitrobot System

Featured cryo-EM sample preparation solutions

Vitrobot System

Vitrification

Improve speed and quality in Cryo-TEM sample preparation with Thermo Scientific Vitrobot System, which provides automated cryo-fixation for fast, easy, and reproducible sample preparation - the first step in obtaining high quality images and repeatable experimental results.

VitroEase Buffer Screening Kit

Cryo-EM sample optimization

Improve your samples for vitrification using the Thermo Scientific VitroEase Buffer Screening Kit, which contains a broad selection of premade cryo-EM compatible buffers and detergents. Combine this easy-to-use kit and the accompanying screening strategy to significantly reduce the time spent on screening cryo-EM grids for high-resolution structure elucidation.

VitroEase Apoferritin Standard

Cryo-EM standards

Reproducibly prepare cryo-EM grids with the homogeneous and stable Thermo Scientific VitroEase Apoferritin Standard. This highly validated pure protein is a quality control sample ideal for verifying microscope status, or for cryo-EM single particle analysis method development.

VitroEase Cryo-EM Training Kit

Vitrification training

Get started preparing your first cryo-EM grids or further hone your vitrification skill with the VitroEase Cryo-EM Training Kit. This kit contains everything needed to get started preparing cryo-EM grids for your Vitrobot System.

Gibco Expi293 Expression System

Protein expression

Protein expression systems, including algae, bacterial, mammalian, insect and yeast, that are easy to use and deliver protein at high yield, suitable for downstream electron microscopy applications.

n-Dodecyl-beta-Maltoside Detergent

Detergents for protein solubilization

Wide selection of detergents available in liquid and solid formats in flexible packaging to support protein extraction from a wide range of sample sources.

Pierce Cell Surface Biotinylation and Isolation Kit

Membrane protein extraction and isolation

Reagents and kits that are optimized for membrane protein isolation and enrichment suitable for a variety of downstream applications.

Pierce agarose resins and columns

Protein purification and isolation

Magnetic beads, magnetic agarose beads, standard and Superflow agarose affinity purification resins, Applied Biosystem POROS resins, and other chromatography media to enable protein purification at nearly any scale.

Zeba Desalting Products

Protein clean-up

Resins, spin columns and devices for contaminant removal or buffer exchange of protein samples.

Gallery proteins

Gallery proteins

Make use of our ready-to-use proteins, like the scFv16 antibody, that will help you stabilize your GPCR/G-Protein complex for structure elucidation by cryo-EM.


Applications

Structural biology research

Structural Biology Research

Cryo-electron microscopy enables the structural analysis of challenging biological targets such as large complexes, flexible species and membrane protein.

Drug discovery

Drug Discovery

Learn how to take advantage of rational drug design for many major drug target classes, leading to best-in-class drugs.

Infectious disease research

Infectious Disease Research

Cryo-EM techniques enable multiscale observations of 3D biological structures in their near-native states, informing faster, more efficient development of therapeutics.

Plant biology research

Plant Biology Research

Fundamental plant biology research is enabled by cryo electron microscopy, which provides information on proteins (with single particle analysis), to their cellular context (with tomography), all the way up to the overall structure of the plant (large volume analysis).


Techniques

Single Particle Analysis

Single particle analysis (SPA) is a cryo-electron microscopy technique that enables structural characterization at near-atomic resolutions, unraveling dynamic biological processes and the structure of biomolecular complexes/assemblies.

Learn more ›

Cryo-Tomography

Cryo-electron tomography (cryo-ET) delivers both structural information about individual proteins as well as their spatial arrangements within the cell. This makes it a truly unique technique and also explains why the method has such an enormous potential for cell biology. Cryo-ET can bridge the gap between light microscopy and near-atomic-resolution techniques like single-particle analysis.

Learn more ›

MicroED

MicroED is an exciting new technique with applications in the structural determination of small molecules and protein. With this method, atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.

Learn more ›

Integrative Structural Biology

To understand protein function, you need complex and structure information beyond individual proteins. Integrative structural biology combines mass spectrometry and cryo EM for the determination of large dynamic complex structure.

Learn more ›

Single Particle Analysis

Single particle analysis (SPA) is a cryo-electron microscopy technique that enables structural characterization at near-atomic resolutions, unraveling dynamic biological processes and the structure of biomolecular complexes/assemblies.

Learn more ›

Cryo-Tomography

Cryo-electron tomography (cryo-ET) delivers both structural information about individual proteins as well as their spatial arrangements within the cell. This makes it a truly unique technique and also explains why the method has such an enormous potential for cell biology. Cryo-ET can bridge the gap between light microscopy and near-atomic-resolution techniques like single-particle analysis.

Learn more ›

MicroED

MicroED is an exciting new technique with applications in the structural determination of small molecules and protein. With this method, atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.

Learn more ›

Integrative Structural Biology

To understand protein function, you need complex and structure information beyond individual proteins. Integrative structural biology combines mass spectrometry and cryo EM for the determination of large dynamic complex structure.

Learn more ›

Samples


Proteins Analysis

Cryo-electron microscopy provides near-atomic resolution 3D protein structure. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context.

Learn more ›


Biopharmaceutical Research

Structural drug discovery is enabled by cryo-electron microscopy, as the method provides near-atomic-resolution detail for small molecules and protein biologics in their fully hydrated state.

Learn more ›


Virus Analysis

Cryo-EM enables the 3D structural visualization of virus particles, and the antigen-antibody interface, at near-atomic resolutions. A virus’s inherent structural symmetry makes it the ideal target for cryo-EM analysis.

Learn more ›


Products

Thermo Scientific Vitrobot vitrification sample system

Vitrobot System

  • Fully automated sample vitrification
  • Blotting device
  • Semi-automated grid transfer
  • High sample throughput
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