Cryo-EM: Freezing Time To Take Snapshots Of Myosin And Other Molecular Systems

Using technologies like electron microscopy (EM) it is possible to capture molecular mechanisms in great detail, but not when these mechanisms are currently moving. The field of cryomicroscopy circumvents this limitation by freezing said mechanism in place using cryogenic fluids. Although initially X-ray crystallography was commonly used, the much more versatile EM is now the standard approach in the form of cryo-EM, with recent advances giving us unprecedented looks at the mechanisms that quite literally make our bodies move.

Myosin-5 working stroke and walking on F-actin. (Credit: Klebl et al., 2024)
Myosin-5 working stroke and walking on F-actin. (Credit: Klebl et al., 2024)

The past years has seen many refinements in cryo-EM, with previously quite manual approaches shifting to microfluidics to increase the time resolution at which a molecular process could be frozen, enabling researchers to for example see the myosin motor proteins go through their motions one step at a time. Research articles on this were published previously, such as by [Ahmet Mentes] and colleagues in 2018 on myosin force sensing to adjust to dynamic loads. More recently, [David P. Klebl] and colleagues published a research article this year on the myosin-5 powerstroke through ATP hydrolysis, using a modified (slower) version of myosin-5. Even so, the freezing has to be done with millisecond accuracy to capture the myosin in the act of priming (pre-powerstroke).

The most amazing thing about cryo-EM is that it allows us to examine processes that used to be the subject of theory and speculation as we had no means to observe the motion and components involved directly. The more we can increase the time resolution on cryo-EM, the more details we can glimpse, whether it’s the functioning of myosins in muscle tissue or inside cells, the folding of proteins, or determining the proteins involved in a range of diseases, such as the role of TDP-43 in amytrophic lateral sclerosis (ALS) in a 2021 study by [Diana Arseni] and colleagues.

As our methods of freezing these biomolecular moments in time improve, so too will our ability to validate theory with observations. Some of these methods combine cryogenic freezing with laser pulses to alternately freeze and resume processes, allowing processes to be recorded in minute detail in sub-millisecond resolution. One big issue that remains yet is that although some of these researchers have even open sourced their cryo-EM methods, commercial vendors have not yet picked up this technology, limiting its reach as researchers have to cobble something together themselves.

Hopefully before long (time-resolved) cryo-EM will be as common as EM is today, to the point where even a hobby laboratory may have one lounging around.

Ptychography Shows Atoms At Amazing Resolution

Cornell University enhanced electron microscopy using a technique known as ptychography in 2018. At the time, it allowed an electron microscope to resolve things three times smaller than previously possible. But that wasn’t enough. The team has now doubled that resolution by improving on their previous work.

The team says that the images are so precise that the only blurring is due to the thermal motion of the atoms themselves. This could mean that you won’t see a further improvement in resolution in the future.

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New Microscope Directly Images Protein Atoms

There’s an old joke that you can’t trust atoms — they make up everything. But until fairly recently, there was no real way to see individual atoms. You could infer things about them using X-ray crystallography or measure their pull on tiny probes using atomic force microscopes, but not take a direct image. Until now. Two laboratories recently used cryo-electron microscopy to directly image atoms in a protein molecule with a resolution of about 1.2 x 10-7 millimeters or 1.2 ångströms. The previous record was 1.54 ångströms.

Recent improvements in electron beam technology helped, as did a device that ensures electrons that strike the sample travel at nearly the same speeds. The latter technique resulted in images so clear, researchers could identify individual hydrogen atoms in the apoferritin molecule and the water surrounding it.

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