Cryo-electron microscopy (cryo-EM) is rapidly becoming the method of choice for biochemistry labs around the world, helping accelerate research. With cryo-EM, molecular details can be seen at biologically relevant resolutions, providing insights into protein function and disease mechanisms, and facilitating effective drug design. Recent advances in instrument design and automation mean cryo-EM is easier to use and even less expensive to obtain. With cryo-EM, you can gain deeper insights into complex biological processes and pathways. While researchers use a variety of techniques to determine protein structure, cryo-EM can provide details on protein function that other methods simply cannot.
Summary of protein structure techniques
Cryo-electron microscopy
In cryo-EM, samples are rapidly frozen (vitrified), preventing the formation of crystalline ice and preserving samples in their natural state. A transmission electron microscope (TEM) is then used to image the sample, capturing a two-dimensional view, or projection, of the specimen. By creating hundreds of projections of the sample from many different angles, the images can be averaged and then recombined into a 3D model of the sample’s structure. Cryo-EM can produce high-resolution images similar to other structural techniques, thanks to high-quality, modern electron detectors and image-processing software.
Nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance spectroscopy (NMR) provides unique information about protein dynamics and interactions, but it is restricted to the atomic-structure determination of small complexes with molecular weights below 40–50 kDa. In NMR, the nuclei of a protein’s atoms interact with a large magnet inside the NMR spectrometer. A series of split-second radio-wave pulses are applied to the sample, which forces the nuclei to resonate at specific frequencies. A complete picture of the protein is developed by combining the measured resonance frequencies.
X-ray crystallography
In X-ray crystallography (XRD), an X-ray beam passes through a small, solid crystal composed of trillions of identical protein molecules. The interaction between the X-ray beam and the sample’s crystal lattice produces a diffraction pattern unique to the protein. Through computer algorithms, the positions and intensities of spots in the diffraction pattern are used to determine the position of each atom in the crystallized molecule. XRD can achieve atomic-resolution for protein samples, including smaller viruses. The visualization of larger samples can be a challenge due to the difficulty in crystallization.
Cryo-EM is filling a gap that is typically unattainable by NMR and X-ray crystallography — allowing the visualization of cells, viruses, and molecular complexes.
Structural biology techniques
Comparison between cryo-EM, X-ray crystallography, and NMR techniques
Cryo-EM
X-ray crystallography
NMR
Sample types
Membrane proteins
Large complex proteins
Ribosomes
Virions
Other macromolecules
Crystallizable samples
Soluble proteins
Proteins with MWS <40-50 kDa
Advantages
Easier sample preparation
Only requires small sample size
Structures are obtained in native state
Works well for broad molecular weight ranges
Easier model building
Obtains 3D structures in solution
Current limitations
Proteins with molecular weights >100 kDa are preferred, but the number of structures from proteins with smaller molecular weights have increased as technology rapidly improves
Crystallization can be difficult and can take months or years to achieve
Solid structure is preferred
Results are in static crystalline state
Diffraction can be difficult
Needs high purity sample
Has a difficult computational simulation
Sample must be isotopically labeled
Sample amounts required
Nanograms to micrograms
Micrograms to milligrams
Micrograms to milligrams
Number of released PDB structures per year
Electron microscopy is the fastest growing technique, quickly becoming the second-most-prolific method.
Adopting cryo-EM
Thermo Scientific products and services can help you adopt cryo-EM quickly and seamlessly, maximizing your return on investment. As the market leader in cryo-EM, we are uniquely positioned to help you and your team be successful at every stage of the adoption process, from financing to guidance on facility requirements, to data processing and analysis.
Financing
We have competitive and flexible financing, leasing, and lease-to own options, as well as resources to assist with grant writing.
The Thermo Scientific Tundra Cryo-TEM is priced to match global grant mechanisms and funding opportunities. We understand that the funding process is still one of the biggest challenges you face when looking to invest in new cryo-EM instrumentation. To reduce some of the stress around this process, we’ve created an Electron Microscopy Funding Support Center where you can find information about available grants, tips for writing funding proposals, and information about financing options specifically for cryo-EM that are offered through Thermo Fisher Financial Services.
Site-preparation services
Environmental engineering experts provide analysis and recommendations, minimizing environmental interference while maximizing system performance.
Delivery and installation
Following installation, our technicians train you to prepare samples and to gather data confidently and safely.
Data collection
Our professionally maintained software solutions provide easy-to-use platforms that focus on automation and user guidance to increase throughput and deliver reproducible results for all user levels.
Adopting a new technology can be daunting. We have created six different service packages that are meant to specifically get new users of the Tundra Cryo-TEM up to speed, ensuring you get the most out of your investment. These services are tailored to reduce the total cost of ownership, while providing all the maintenance, training, and support your lab will need. In addition to custom packages for the Tundra Cryo-TEM, we offer a variety of service packages to ensure that anyone buying a cryo-TEM gets the most out of their investment.
Single particle analysis is a cryo-EM technique that enables structural characterization at near-atomic resolutions, unraveling dynamic biological processes and the structures of biomolecular complexes or assemblies.
Sample preparation
Vitrification
Screening
Data acquisition
Structure visualization
High-quality single particle analysis starts with thorough sample preparation and screening. A variety of traditional sample preparation techniques can be used, including negative-stain screening and chromatography. Once the aqueous sample is sufficiently purified, it is rapidly frozen to suspend the specimens in a layer of amorphous (vitreous) ice (vitrification). By avoiding ice crystallization, the samples are preserved in a near-native state, essentially taking a snapshot of their structures in solution. To find the optimal areas of ice for data collection, qualitative screening of the sample frozen atop an EM grid is needed. Data collection consists of high-resolution imaging with a TEM specifically designed for cryo-applications. With advances in data collection software, individual particles can be automatically identified in the TEM image and grouped according to particle orientation, a process that is simplified and accelerated by robust, reliable automation. Once sufficient particle data is collected the data can be recombined into a 3D representation of the protein or protein complex.
Microcrystal electron diffraction (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.
Sample preparation
Vitrification
TEM low-dose screening
Data collection
Reconstruction
High-resolution structural determination of small molecules and proteins by MicroED starts with sample crystallization using the same methods that are found in X-ray crystallography. However, much smaller crystals (~100 nm in size) can be used in MicroED because crystals interact more strongly with electrons than they do with X-rays. This may significantly shorten the sample preparation process and allow the analysis of crystals that are too small to diffract with other methods. Data is then acquired on a cryo-TEM, using electrons as the incident beam. Data collection is completed in only a few minutes, and 3D structures can be determined at atomic resolution.
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, with an enormous potential for cell biology. Cryo-ET can bridge the gap between light microscopy and near-atomic-resolution techniques like single-particle analysis.
Cell culture
Vitrification
Localization by fluorescence
Thinning by milling
Imaging by TEM
Reconstruction and visualization
Cryo-electron tomography (cryo-ET) provides label-free, fixation-free, nanometer-scale imaging of a cell’s interior in 3D and visualizes protein complexes within their physiological environments. Using a correlative light and electron microscopy approach allows tagged proteins to be targeted by fluorescence microscopy before subsequent higher-resolution imaging with cryo-EM. Many cells are too thick for cryo-ET, so the vitrified cells must be thinned with a cryo-focused ion beam (cryo-FIB) prior to imaging in a TEM.
Cryo-EM is accessible to a broader range of scientists than ever before, including first-time users in the fields of biochemistry, molecular biology, structural biology, cell biology, and more. Watch these videos to see how our customers are using electron microscopy to advance their research.
Prof. Erica Ollmann Saphire (La Jolla Institute for Immunology) discusses how cryo-EM has allowed her team to understand a range of virus structures from Ebola to SARS-CoV-2, leading to insights for drug and vaccine development.
Dr. Tamir Gonen, the developer of MicroED, discusses his current research at UCLA, which focuses on the structures and functions of medically important membrane proteins that are involved in homeostasis.
Professor Stefan Raunser and his team discuss their lab’s expansion from single particle analysis with cryo-EM to cryo-ET for skeletal muscle research. Together they share how this technique dramatically increases their ability to not only study ensembles of protein complexes but to better understand how they interact natively within a cell.
Discover how Cryo-EM is used to advance all areas of the life sciences. Explore some of our featured eBooks and start your journey today.
Untangling neurodegeneration
Recent developments in cryo-EM facilitate profiling of the large filaments and plaques that are common to neurodegenerative diseases. Learn how cryo-EM helps classify tauopathies.
Cryo-EM helps researchers overcome challenges in virus purification and protein homogeneity. Read our new eBook to find out how cryo-EM revolutionizes antiviral drug discovery and drug design.
Visualize how cancer therapeutic agents interact with receptors
Leverage structural insights to better understand the conditions for cancer cell growth and identify new ways to treat cancer. Discover how cryo-EM is revolutionizing cancer research.
Exploring structure–function relationships with cryo-ET
Access the inner workings of cells in 3D at unprecedented nanoscale resolution using cryo-ET. Read more about cryo-ET and current scientific insights that use this technique.
* Based on best published performance, actual results will depend on non-microscope factors such as sample and user experience. Not a promise of biological resolution performance.
Looking to find a cryo-EM facility near you?
There are nearly 300 world-class cryo-EM facilities where you can advance your research by obtaining cryo-EM structures and proof of concept data using your own samples. Most facilities have an application form that will guide you through proposal requirements. Although these vary by facility, generally you can expect to submit a research proposal for independent scientific and technical evaluation based on:
Scientific background and project significance
Technical suitability and feasibility, including descriptions of the sample and sample quality
Individual facilities also offer different levels of service, which may include sample preparation, grid screening, and cryo-EM training.
Fill out the form below to speak to one of our scientific experts about how cryo-EM can accelerate your scientific discovery and what microscope is best suited for your lab or institution.
Electron microscopy services for the life sciences
To ensure optimal system performance, we provide you access to a world-class network of field service experts, technical support, and certified spare parts.
Epitope mapping <5 Å
H2O molecules and model building <2.5 Å
<2 Å H atoms, ligand identification, interaction
