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Cryo-electron Microscopy – photographing molecules as they are

Manidipa Banerjee & Krishanu Ray

The Nobel Prize of 2017 is jointly awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for their contribution in developing the technique of Cryo-electron Microscopy for imaging large molecules in vitrified ice, which is considered as if they are in their natural environment. Of course, this is a big deal because it opens the window of seeing the biological molecules such as proteins in their natural environment. My objective here is to deconstruct the history and usefulness of this method at the popular level. Also, highlight its impact on future research and development of new applications.

Let us first go back to the history. Microscopy as a tool has ushered in many revolutions in science and technology which further enhanced the quality of human life. It has also majorly influenced human belief system. Imagine how one could accept the fact that malaria and leprosy are caused by tiny organisms called pathogen if they were never seen. Because the human mind is strongly oriented towards accepting what is seen, we tend to believe the most once we see something. Although the practice of science over several centuries have generated scores of ways to infer what is unseen through many indirect ways and logical deductions, the influence of visual confirmation has maintained the prime place amongst all these. Thus, microscopy and various forms of image gathering have always captured our attention by enabling us to see the unseen. Modern microscopy has come a long way from its debut with Anton van Leeuwenhoek’s magnifiers in late 17th century. Initially, the microscopy was limited to enlarging the objects with innate, optical contrast-defined structures such as cell wall, microorganisms, etc. The invention of dye staining and improvements in the compound microscope designs with artificial lighting arrangements or illumination systems created the field of histology and histopathology. The later, greatly influenced development of the current diagnostic practices in medical science and treatment of diseases; then came the era of advanced fluorescence spectroscopy and its convergence with microscopy, such as confocal microscopes, made it possible to ‘see’ molecules in action. It also improved our understanding of cellular physiology, and subsequently, helped the discovery of many more modern medicines. However, a gap remained, i.e., none of these technologies can actually ‘show’ where a particular drug binds.

It was the preserve of crystallography. Since the discovery of X-ray diffraction technique by the father-son team of Braggs in the early 20th century, the X-ray crystallography has made a significant inroad in enabling us to see the atoms as they are within a molecule and helped find several cures. Notwithstanding the centrality of this technique in modern medicine, the images obtained by the X-ray diffraction technique can only be considered as an average view of reality or a deduction. It is also hamstrung by the fact the one needs a large number of the same molecule to be packed in an ordered array for obtaining such a deduction. In most cases, the molecules of life work in tandem with many other different kinds. Their ‘structures,’ as well as ‘function,’ are more often determined by the company they keep.

The Cryo-electron microscopy has the potential to provide a breakthrough in this scenario. It can enable visualization of individual molecules at near-atomic details in a natural setting because it does not require packing the molecules in a crystalline array. Therefore, one can potentially ‘see’ how TB bacteria evade the most potent drug ever invented or find an inescapable lock preventing their invasion into our cells.

How does one get this information? Let us now get into some detail behind this invention. It was already proven in 1930’s that when electrons are accelerated to nearly reaching the speed of light, they behave as a wave. This particle-wave duality was demonstrated in the most obvious way by Max Knoll and Ernst Ruska at the Berlin Technische Hochschule in 1931 by installing the first ever electron microscope. The instrument showed details within a material that were hitherto remained ‘unseen’ by using visible light. Biologists, material scientists, and medical sciences immediately understood the power of this technique and its usefulness in applications. Since then, electron microscope has been used as a tool for critical pathological analysis of tissue material and in cell biology research. There was, however, one major impediment. The electrons deflect better from the higher-Z, or larger atoms and biology is mostly managed by Hydrogen, Carbon, Oxygen and Nitrogen-based molecules. Also, illuminating a specimen with too many electrons at a time drills a hole by vaporizing the material. Therefore, the electron images are always very faint. Hence, one needs to keep the specimen steady for a ‘long’ time to get a clear, sharp, image. Further, the electron beam must travel in high vacuum lest they will be diverted or absorbed by the air, and all biological systems are full of water, which will evaporate as soon as one puts them in a vacuum.

The Achilles heel of biological electron microscopy was bypassed through systematic replacement of water in biomaterial with resins and making ultrathin slices. They are also heavily doped with Osmium, Lead, and Uranium. All these treatments altered a majority of natural organization of molecules. So we got an image of a highly manicured artifact. It is comparable to the images projected on celluloid after applying heavy makeup and dressings on the artists/subjects. Nonetheless, the information obtained was highly valuable and made innumerable vital discoveries, such as the real cause of Scrappy, the Alzheimer’s disease and much more.

Still, the desire to ‘see the molecule as they are’ was nursed by many. The first breakthrough came when Jacques Dubochet demonstrated that one could render ice invisible under an electron beam by freezing a thin layer of water so quickly that it could not crystallize. By keeping the ice in this ‘vitreous’ form on a stage inside the electron microscope, one could potentially image items that are dissolved in this ice. The technique was further improved by Richard Henderson and his colleagues. They also introduced biological macromolecules into this vitreous ice and looked at them under the electron microscope. The results were stunning, almost magical. Suddenly, the prospect of looking at a microtubule or ribosome became a reality. Still, there was a major drawback, i.e., how to extract the atomic structure from the blobs seen on the image plates. Physicist-turned-Biologist, Joachim Frank, introduced the deduction which made it possible. He suggested that by collecting a large number of images at multiple angles, classifying these images according to certain geometric criteria and making class-averaged images, one could remove the ‘noise’ from the blobs and make them look better. More importantly, one could extract the three-dimensional topology of individual molecules using this method. The slow collection of images using photographic films was a major impediment that prevented full realization of this technique as an industry-grade tool. The advent of ever so fast camera chips and image analysis technique combined with computational powers kept improving the prospect until we got the direct electron-detecting camera chip. With the ability to ‘see’ the electron image without having to convert them to an optical form first not only improved the resolution, but it also managed to capture events occurring at sub-millisecond speed. One can now literally see how a ribosome makes protein in real time.

So what! One may still ask what’s the big deal. Here is a riposte. Since the beginning of evolution, organisms made electricity from sunlight and used it to convert carbon dioxide to glucose – the primary food source of life. Even though humanity also has invented ways to make electricity from sunlight using silicon wafers, the efficiency is very low. Moreover, we still can’t make glucose from thin air and sunlight. The Cryo-electron microscopy extends the promise to show how it happens in reality within a chloroplast or on the membrane of cyanobacteria. The race had already begun decades ago to unlock the secret using this particular tool. The ability to see how a drug binds to a ribosome or on a channel protein at the surface of a bacterium or a cell would help to develop a more useful therapy. It could also revolutionize the development of new application materials using organics.

One can go on with many more examples, but I guess you are already exhausted reading this rant. So with wishes and optimism of a great ‘cryogenic’ era, I must now withdraw.

Additional reading:
1. Nature vol 550, PP-167 (12 October 2017) doi:10.1038/nature.2017.22738
2. Nature vol 525, PP 172–174 (10 September 2015) doi:10.1038/525172a

Cryo-Electron Microscopy in India: Cryo-Electron Microscopy has been practiced in India from 2000 onwards. The first sets of systems were installed in NIV Pune, IIT-B Powai, CCMB Hyderabad and NICED Kolkata around the same time. This was followed by an installation in TIFR, Mumbai, IIT-Delhi and several other places. Most of these were low energy systems good for low-resolution contrast imaging. Prof. Amar Nath Ghosh and his student Somnath Dutta at NICED produced the first 3D-Cryo-electron microscope structure of a bacterial protein (Vibrio cholerae hemolysin oligomer) at NICED in 2010. The use of the more high energy Cryo-Electron microscopy started with the installation of a 300 KeV system at CSIR-IICB, Kolkata. Recently, a state of the art system is installed in NCBS-TIFR, Bengaluru. With this installation, active and high-resolution analysis using the technology can start in this country. Interestingly, several young and enthusiastic faculty have joined in the last few years and started their research using the Cryo-electron microscope (see the list below), and several structural biologists who have been using X-ray crystallography as the main tool is contemplating to switch to the Cryo-TEM. Therefore, the prospect of future research in India using this technology can only improve.

Experts practicing Cryo-Electron microscopy in India:

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Local cryoEM Expertise: A state-of-the-art facility for cryoelectron microscopy, consisting of a 300 KV cryoelectron microscope equipped with the direct detector and a phase plate, is currently being set up in NCBS, Bangalore. It is expected to cater towards data collection requirements of the Indian structural biology community. There are also several 200 KeV microscopes, which are either currently functional or are being installed or purchased for several institutes in the country. These can potentially serve as screening microscopes in the initial part of the workflow for cryoelectron microscopy and single particle reconstructions – such as determining freezing conditions for different samples, or for low to medium resolution model generation. It is hoped that access to more state-of-the-art facilities will aid the efforts of local laboratories to effectively utilize this technique to address scientific questions.

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CryoEM in Virology: Cryoelectron microscopy and single particle reconstructions, as well as cryotomography methods, are particularly essential for virology research. Viruses are obligatory intracellular pathogens that interact with cellular components to gain entry into host cells, where they carry out replication and generation of progeny virions for the perpetuation of the infection. Structural detail of the conformational alterations in viruses during entry, assembly, and disassembly, interaction with receptors and other cellular components or with cellular membranes can be studied efficiently in using cryoelectron microscopy and cryotomography. Due to the dynamic nature of these processes and the large size of complexes formed, the other available structure determination techniques like X-ray crystallography or NMR are either not suitable or fairly challenging for understanding these processes. Cryoelectron microscopy based reconstructions are also very essential in determining structures of new or re-emerging pathogens quickly (example: Zika Virus) to aid the drug discovery process.

Authors’ profile:
Manidipa Banerjee, Indian Institute of Technology, New Delhi, India
Krishanu Ray, Tata Institute of Fundamental Research, Mumbai 400005, India