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Electronic cryomicroscopy, a Nobel-winning tool to image molecules in high resolution

Last October 4, the Royal Swedish Academy awarded the Nobel Prize for Chemistry to Jacques Dubochet, Joachim Frank and Richard Henderson for the development of electronic cryomicroscopy. The technique, a tool offered by departments such as that of the University of Barcelona, was chosen “method of the year” by Nature Methods magazine in 2015. “One of the great advantages of electronic cryomicroscopy over the other two structural methods, X-ray diffraction and Nuclear Magnetic Resonance (NMR) is its capacity to gather high-resolution structural information on macromolecular complexes that are transient, and therefore, unstable,” says José María Valpuesta, scientist at the National Center for Biotechnology (CNB-CSIC) in remarks to Biocores.

Those characteristics of transiency and instability are a serious problem for the X-ray diffraction and Nuclear Magnetic Resonance methods. The first case requires the crystallization of the complexes, which is not always possible, while in the second, it is difficult to resolve protein structures or large protein complexes. Electronic cryomicroscopy, on the other hand, can gather high-resolution information without having to first obtain crystals, on samples that cover a vast range of sizes, from approximately 100 kilodaltons to several megadaltons.

Source: Adam Baker (Flickr)

Another advantage is that this method requires a much smaller sample and fewer modifications, as Rafael Fernández-Leiro, scientist at Spanish National Cancer Research Center (CNIO) explains to Biocores. “The possibility of using small sample quantities opens the door to hard-to-obtain samples, due to their difficulty during the purification process or because there is little sample to start with, such as native samples or those of patients,” Fernández-Leiro says. The CNIO researcher, who applies cryomicroscopy in the design of new medications against cancer, also highlights its capacity “to use samples with a higher level of heterogeneity,” which facilitates analysis of dynamic systems with transient interactions.

Membrane proteins, which are also one of the protein groups with the most therapeutic targets, is one of those that has most benefited from the advancements in microscopy, as their study by crystallography was extremely complex,” says Fernández-Leiro. Research into these proteins has revealed the protein complexes responsible for DNA replication and repair, systems that are “extremely dynamic, and have highly transient interactions,” which makes the samples heterogeneous. The CNIO scientist has managed to resolve their structures in multiple forms, with the aim of “understanding in detail how they work and how they are regulated,” two results that other structural methods had been unable to achieve. 

Biomolecular CryoEM workflow
Source: Reichow Lab, Portland State University

On another note, Valpuesta uses electronic cryomicroscopy in the study of molecular chaperones. In one of their recent studies, published in Nature Structural & Molecular Biology, his team used this technique to determine how Hsp70 chaperones operate, generating stress over other molecules through “collisions” and “pulling” to transport them or disrupt the associations between them. The CNB-CSIC scientist tells Biocores that the most important results achieved with electronic cryomicroscopy have been to “gather structural information on how chaperones interact, not just with their substrates, but also other chaperones and proteins that assist them in processes of folding and protein degradation.”

In addition to the applications developed by these researchers, scientific groups from around the world have used this tool to gather ground-breaking structural data, studying large macromolecular complexes such as ribosomes, various RNA polymerases, spliceosomes, viruses, dyneins, or analyze the previously-mentioned membrane proteins, such as receptors or ion channels. “For example, a spectacular recent study looked into the protein fibers that accumulate in the brains of Alzheimer’s patients, directly from a sample of patients. By doing so, scientists could observe the true structure of the fibers involved in the disease,” says the CNIO’s researcher. Fernández-Leiro is referring to a study published in Nature in which researchers discerned the structures of the tau protein filaments that accumulate in patients who have this neurodegenerative disease.

Cryo-EM structure of haemoglobin
Source: Danev et al., Nature Communications 2017.

In the opinion of Fernández-Leiro, the most important challenge for electronic cryomicroscopy is to “routinely” reach resolutions below 2Å. Both researchers agree that, thanks to advancements in instrumentation, they will reach truly atomic resolutions in coming years. The CNIO scientist points to other major challenges in using this technology. The first is the throughput, as “recording a structure still takes a number of days in the best-case scenario,” says Fernández-Leiro. The second limitation has to do with the size of the molecules; the smallest protein imaged by electronic cryomicroscopy is hemoglobin, at 64 kDa, a result recently published in Nature Communications. Nonetheless, proteins below 100 kDa are “still extremely complicated”, claims Fernández-Leiro. Through technological improvements, scientists will also be able to overcome this limit, and thus be able to “photograph” the most unknown side of the molecules of life.