Structure-based designs facilitate drug discovery, and cryogenic electron microscopy (cryo-EM) is an increasingly important tool to determine high-resolution structures of proteins and protein complexes. This is especially true for certain classes of proteins that have proven difficult to crystallize.

The promise and versatility of cryo-EM in structural biology resulted in a 2017 Nobel Prize in Chemistry that was awarded jointly to Jacques Dubochet, PhD, Joachim Frank, PhD, and Richard Henderson, PhD, for “developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.”1

In cryo-EM, as in other technologies, sample preparation is the key to avoiding the “junk in, junk out” phenomenon. Indeed, in cryo-EM, sample preparation has been a primary bottleneck. The process appears straightforward, but the biomolecules disagree. They can move, denature, aggregate, or preferentially orientate, affecting resultant data. A better designed sample grid could help tame the biomolecules’ behavior.

Inventive researchers are trying to democratize cryo-EM, a technology that can be inaccessible because its cost is nontrivial. Top-of-the-line cryo-EM instruments operate at voltages of 300 keV and cost about $5–7 million. Less powerful instruments are being developed that operate at around 100 keV. They cost about $1–2 million.2

Researchers working on low-power, low-cost instruments include scientists in the Structural Studies Division of the MRC Laboratory of Molecular Biology at Cambridge University. The division’s group leader, Christopher J. Russo, PhD, is the senior author of a recent paper that described the potential advantages of 100 keV instruments.3 “Reducing the electron energy … offers both cost savings and potentially improved imaging,” the article noted. “The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage.”

Recent advances, lingering challenges

Initially, cryo-EM produced only fuzzy images of biomolecules, limiting its applications in structural biology. According to Robert Thorne, PhD, professor of physics, Cornell University, and founder and chief technical officer, MiTeGen, the resolution was high enough to resolve the overall shape and, perhaps, the secondary structure—but not the atoms.

“That changed a decade ago with the development of more efficient direct electron detectors and phase plates and good data processing software,” Thorne points out. Now it is quite common to get near atomic resolution of 3–4 Å, or even 2.5 Å.

Many biological systems, such as membrane proteins, have proven difficult to crystallize, and so determining their structures using X-ray crystallography has not been possible. Cryo-EM does not require crystals. Importantly, the technology can determine structures of large complexes made up of multiple proteins and of proteins or complexes with biologically relevant heterogeneity. X-ray crystallography still gets higher resolution on average, but crystallization eliminates most heterogeneity.

The technology is versatile. Thorne has been impressed by the cleverness with which people have applied cryo-EM to solve challenging problems.

But each cryo-EM application starts with a good sample, and cryo-EM sample preparation is the primary bottleneck to getting good data. “It is remarkable that the crude methods most widely used work,” Thorne observes. “It is always complicated and sample specific.” He adds that it is necessary to get a thin film of glassy ice that has the right thickness and contains randomly oriented intact molecules that are in biologically relevant configurations.

For example, biomolecules tend to find their way to air–water interfaces very quickly, and at these interfaces, some complexes can fall apart. Biomolecules can denature, partially unfold, aggregate, or become preferentially oriented. Cryo-EM relies on having images of the particle in all possible orientations. To prevent biomolecule movement during sample preparation and image acquisition, cryo-EM systems use grids that have been coated with graphene and modified with protein-binding functional groups such as amines, thiols, and carboxylic acids.

Functionalized grids and many other cryo-EM tools have become available to cryo-EM users. Such tools, MiTeGen indicates, can measurably improve the ease, reproducibility, and quality of cryo-EM experiments. The company adds that it engineers, manufactures, and distributes products not just for cryo-EM experiments, but also for crystallization, crystal harvesting, cryocooling, and X-ray diffraction data collection of proteins, viruses, and small molecule/inorganic compounds.

Addressing sample preparation issues

Manual cryo-EM sample preparation can be problematic. A few microliters of sample are applied to a disc-shaped piece of metal mesh that is about 3 mm in diameter and made of malleable copper or gold, and blotting paper is used to draw the liquid into the supporting layer to form a thin liquid film. Then the construct is plunged into liquid ethane, vitrifying the sample.

“The technique can take months to master and has a lot of variability, in particular the blotting time and contact angle,” says Paul Thaw, product manager, integrative structural biology, SPT Labtech. “More important, a good grid is confirmed only after it is in the microscope.” SPT Labtech’s next-generation chameleon instrument is designed to automate the sample preparation process. According to the company, chameleon can vastly reduce the variability caused by manual handling.

A camera-monitored robot aspirates a few microliters of sample into a humidity zone, and the sample is rotated while a grid specially designed to eliminate the blotting process is prepared. “Nanowires on the bars draw the fluid into the grid, speeding up the process,” Thaw details. A hydrophilic charge is applied to the grid and then the sample is applied. An algorithm is used to evaluate the liquid behavior and ensure that the desired thickness of liquid is achieved. Then a robot plunges the grid into liquid ethane.

“Due to running costs, you want electron microscopes operating at the maximum capacity and output,” Thaw explains. “The chameleon provides consistency and video feedback on likely grid quality. The time lapse between when the sample hits the grid and is frozen can be as little as 54 milliseconds, orders of magnitude faster than traditional standard manual plungers.”

In cryo-EM, molecules are flash frozen in a glassy sheet of water and bombarded by electrons. Multiple images of the molecules within the ice are captured (left). Because these images are blurry, data analysis software is used to process them by the tens of thousands to generate near-atomic-resolution structures (right). In a recent study from Cornell University, it was determined that if liquid nitrogen were used rather than the usual coolant, liquid ethane, sharper images could be produced.

Using a faster plunge-freezing in single-particle cryo-EM can reduce unwanted phenomena at the air–water interface, phenomena such as preferred orientation or dissociation. Indeed, this possibility was discussed in a recent article that described experiments undertaken with the chameleon and other instruments. The article noted that in a typical cryo-EM grid preparation, both sides of the thin film are exposed to the air–water interface, which can be a hostile environment for proteins and macromolecular complexes. The article also presented evidence that “higher grid-making speeds,” such as those enabled by the chameleon, can help investigators “reduce particle damage and subunit dissociation.”4

The chameleon’s grid-making speed recently facilitated a study of a complex protein-folding process, namely, the GroEL-GroES chaperonin cycle. Faster plunge-freezing reduced denaturation at the air–water interface and improved the orientation distribution of particles, permitting a more detailed view of the chaperonin-assisted folding pathway and mechanism. Detailed results from the study appeared in a recent preprint. The preprint’s authors reported, “Our cryo-EM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.”5

Another point emphasized by Thaw is the overall crucial importance of the sample support in cryo-EM. He points out that new developments, such as HexAuFoil (a gold hexagonal grid with holey support film developed by Russo’s group), will crucially improve data quality for a large majority of samples.6

The chameleon instrument from SPT Labtech is designed to automate sample preparation workflows in cryo-EM microscopy. Key features of the instrument include high-speed blot-free vitrification, automated grid handling, and the ability to screen grids based on likely ice thickness. Guided software protocols can help users manage experimental frameworks, limit manual interventions, and obtain more consistent results.

Expanding real-time data analysis

“Cryo-EM is making incredible headway in structural determination and drug discovery for debilitating neurodegenerative disorders—Alzheimer’s, Parkinson’s, and Huntington’s diseases—as well as for cancer,” says Edward Pryor, PhD, director, product management, Thermo Fisher Scientific.

Thermo Fisher Scientific provides streamlined end-to-end workflows across a variety of cryogenic transmission electron microscopy (cryo-TEM) methods, including single-particle analysis, microcrystal electron diffraction, and cryo-
electron tomography. The cryo-TEM portfolio includes the Tundra cryo-TEM and the latest generation Glacios and Krios cryo-TEMs. To facilitate cryo-electron tomography sample preparation, Thermo Fisher Scientific has cryo-FIB (focused ion beam) and cryo-PFIB (plasma focused ion beam) systems, including the Aquilos 2 Cryo-FIB, the Helios Hydra PFIB, and the Arctis Cryo-PFIB, which was recently awarded a 2023 Microscopy Today Innovation Award.

“With the Tundra,” Pryor asserts, “researchers can routinely obtain structures at biologically relevant resolutions of 3–4 Å and prepare high-quality samples for further analysis on the Krios or Glacios cryo-TEMs.”

Designed for streamlined sample optimization and routine data collection, the Glacios 2 cryo-TEM is configurable for single-particle analysis, cryo-electron tomography, and microcrystal electron diffraction. Samples can be directly transferred to the Krios cryo-TEM. The ultra-high-resolution Krios G4 cryo-TEM, also configurable for all three applications, can be paired with Thermo Fisher Scientific’s E-CFEG cold field emission gun, Selectris imaging filter, and Falcon 4i detector for rapid <2 Å resolution data acquisition.

Microscopy and Microanalysis 2022
At Microscopy and Microanalysis 2022, Thermo Fisher Scientific and Structura Biotechnology announced a collaboration to introduce a new software, Embedded CryoSPARC Live. It is a single-particle analysis solution that can enable cryo-EM users to obtain high-quality data while reducing the time needed to transform raw data into high-quality 3D protein structures. Instead of taking days, structures can be available in hours. The software is designed to integrate with Thermo Fisher’s Smart EPU software and cryo-TEM technology.

Pryor notes that each step in the single-particle cryo-EM workflow poses unique challenges. For instance, in the data acquisition step, advancements in software such as Thermo Fisher’s Smart EPU have increased ease of use through advanced automation and the integration of artificial intelligence. However, many users continue to rely on human expertise to evaluate whether data acquired are suitable for high-resolution structure determination.

Thermo Fisher’s collaboration with Structura Biotechnology offers another slant for single particle cryo-EM data acquisition. The companies are working together to develop a new single-particle analysis software solution called Embedded CryoSPARC Live. It integrates a version of Structura’s CryoSPARC Live with Thermo Fisher’s Smart EPU software and cryo-TEM technology.

“Our approach,” says Ali Punjani, PhD, co-founder and CEO, Structura Biotechnology, “enables real-time cryo-EM data analysis, allowing users of all experience levels to obtain high-quality data while reducing the time, from days to hours, that it takes to transform raw data into high-quality 3D protein structures.”

Democratizing the technology

In cryo-EM, as in other fields, reviewing the basics can reap rewards. For example, when Russo and his colleagues at the MRC Laboratory of Molecular Biology were studying how to achieve the dual goal of optimizing the amount of useful electron scattering off a protein’s atoms and minimizing the amount of radiation damage, they discovered, to their surprise, that the optimum voltage for single-particle structure work is around 100 keV, not 300 keV.7

Their next step was to build a prototype. They reduced the voltage of a standard, commercial 200 keV field-emission gun (FEG) microscope to 100 keV and used a side-entry cryoholder. Then they attached a commercial hybrid pixel camera designed for X-ray detection to the camera chamber for low-dose data collection. Five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein, and hemoglobin. The data sets corresponding to these specimens were used to reconstruct 3D structures with resolutions between 3.4 and 8.4 Å.3

With that success, the motivated team moved forward and built a dedicated modular FEG operational to 100 keV. The present design lends itself to integration with existing high-voltage electron columns. Possibilities include upgrading thermionically operated TEMs or electron beam lithography systems into FEG operation to advance research and development applications.8


1. Nobel Prize Outreach. The Nobel Prize in Chemistry 2017. Published October 4, 2017. Accessed August 2, 2023.
2. Hand E. Cheap shots. Science. 2020; 367(6476): 354–358. DOI: 10.1126/science.367.6476.354.
3. Naydenova K, McMullan G, Peet MJ, et al. CryoEM at 100 keV: a demonstration and prospects. IUCrJ. 2019; 6(Pt 6): 1086–1098. DOI: 10.1107/S2052252519012612.
4. Gardner S, Darrow MC, Lukyanova N, et al. Structural basis of substrate progression through the chaperonin cycle. bioRxiv. Posted May 31, 2023. DOI: 10.1101/2023.05.29.542693.
5. Klebl DP, Gravett MSC, Kontziampasis D, et al. Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales. Structure. 2020; 28(11): 1238–1248.e4. DOI: 10.1016/j.str.2020.07.018.
6. Naydenova K, Jia P, Russo CJ. Cryo-EM with sub-1 Å specimen movement. Science. 2020; 370(6513): 223–226. DOI: 10.1126/science.abb7927.
7. Peplow M. Cryo-Electron Microscopy Reaches Resolution Milestone. ACS Cent Sci. 2020; 6(8): 1274–1277. DOI: 10.1021/acscentsci.0c01048.
8. El-Gomati M, Wells T, Zha X, et al. A modular 100 keV vacuum sealed FEG for high-resolution electron microscopy. Microsc Microanal. 2021; 27(S1): 846–847. DOI: 10.1017/S1431927621003317.


Previous articleLiquid Biopsy Technologies Hasten Progress in Precision Oncology
Next articleNonviral Platforms Streamline Gene Therapy Delivery