Determining Protein Structure with Cryo-EM

Recent developments in cryo-electron microscopy (cryo-EM) have made the technology faster, easier, and more accessible, with its use facilitating discoveries in fields from structural biology to drug discovery. But despite its now widespread use, there remain a number of challenges associated with the technology. In this article, we present some tips that can help alleviate these problems and make cryo-EM a powerful part of your toolkit for protein structure determination.

Applications

Extensive use of cryo-EM has led to substantial knowledge gain in many fields. One such area is in gene expression and regulation where cryo-EM has been used to help resolve the structure of the spliceosome. Important breakthroughs have also been made in chromatin remodeling, including determination of the structure of the Snf2-nucleosome complex in different nucleotide states. Additional details about protein synthesis and degradation are also being revealed with cryo-EM, including insights into the human mitochondrial ribosome and the structure of the human 26S proteasome.

The field of membrane proteins is another area where cryo-EM is really coming into its own, bringing new insights by elucidating the structures of proteins difficult to resolve with x-ray crystallography. And with an estimated 60% of drugs targets being membrane proteins, this is a major step forward. Many ion channels and transporters have also been resolved, including the human cholesterol transporter NPC1 and working mechanisms of voltage-gated Ca2+ and Na+ channels. Resolution of the respirasome respiratory chain super-complex has also been achieved—holding important potential for the treatment of cellular respiratory-related diseases.

Developments are also being made in the structure of viruses, helping to elucidate both the mechanisms of assembly and infection, thereby aiding in drug design. The structure of the nucleocapsid of herpes simplex virus type 2 (HSV-2), for instance, revealed the mechanism of human herpesvirus genome packaging—helping in the control and prevention of a variety of diseases caused by the virus. The structure of neurodegenerative disease and innate immune system related proteins are also a major area of interest. The structure of the human γ-secretase for example, has provided insight into the pathogenesis of Alzheimer's disease.

Challenges

But while important breakthroughs are being made with cryo-EM, a number of challenges with the technology still exist. And for many, the high cost of the equipment means that even getting started is a challenge, although the continued development of shared or centralized facilities should help to increase access.

Once started, one of the biggest disadvantages remains the low signal to noise ratio—reducing image quality and making it hard for users, particularly new users, to identify what they are seeing. Studying proteins in their native physiological states also remains a challenge, as does tilt imaging—the cross section of the frozen sample making it difficult to complete.

But with cryo-EM, we cannot mention challenges without discussing sample preparation. This is by far the most challenging and time-consuming stage. And it will remain so until sample preparation can be effectively automated.

Tips when using cryo-EM

Sample preparation

Sample preparation is tough—but stick with it, time spent here will pay off in image quality later. Sample purity is key. You need a pure sample. Going through multiple stages of purification is essential: affinity chromatography, SDS-PAGE, size-exclusive chromatography, ion-exchange, etc. Try them all, and then keep trying them until your sample is super pure. Then maybe purify some more for good measure! You should also use a low-salt, organic-substance free buffer for preparation, with additives reducing sample contrast and image quality. Negative stain electron microscopy can be used to check your sample prior to imaging. If this is not clear, you need to purify again before moving on. And remember, good sample preparation is a challenge; much better to repeat your preparation now and prevent any future disappointment.

Grid choice and freezing

Choosing the right grid or mesh support for your sample is also essential to your success. There are many different types of grids available and while choosing the right one may come down to trial-and-error, looking at what other scientists have used for similar samples can substantially speed up the process.

Taking time to optimize your grid preparation is next on the list. Ice thickness and vitrification are key. Thick ice increases electron scattering and reduces image quality. Following grid mounting, samples must be rapidly subjected to plunge freezing—dipping the grid in liquid ethane to rapidly cool to cryogenic temperatures. You should then ensure you administer the grid to the microscope without warming or coming into contact with air.

Hone your skills and ask for help!

Learning the subtleties of cryo-EM takes time, with new users needing to learn the complexities of sample preparation, as well as reading image quality and identifying artefacts. While these skills can be taught, experience counts for a lot, so hang in there. Sharing knowledge is also important. Cryo-EM is still a developing field and the whole community is learning all the time. So, share your knowledge and work with others—new discoveries in the field are a benefit to everyone.

Cryo-EM is a powerful tool and one that is increasingly used—despite the challenges that remain. But with the technology continually improving and shared tips helping enable impactful use, cryo-EM can be an invaluable, complimentary weapon in the fight to resolve protein structures. And remember, effective cryo-EM comes with experience, essential time investment, and knowledge sharing.