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An engagement with the resolution revolution

Vinoth Kumar Kutti Ragunath

I had never looked at an Electron microscope until the start of my PhD when I used one. It was during the Christmas of the year 2000 that I left India to start my PhD at the Max-Planck1303_2013-05-7KriosI (1) Institute of Bio-physics in Frankfurt under Prof. Werner Kuehlbrandt. I had not realized that time it was holiday in Europe and I spent my initial period getting used to the cold and white Christmas along with reading on electron microscopy and membrane proteins and more importantly learning how to cook (a downside of being a vegetarian in Europe). Just after the New Year, when everybody was back in the lab, I was taken down to the electron microscopes in the basement. There were several of them each occupying a room, some small ones and others very big and then realized that they bring me closer to the atomic details of proteins. I was very excited to be learning something new and at the same time had the feeling that both EM and membrane proteins are the fields that one can spend entire scientific career.

IMG_1386Electron microscope (EM) is an imaging technique that uses high voltage electron beam and has the power to visualize atomic details of materials. Since the wavelength of the electrons is very small, EM is superior to light microscopes in their ability to resolve details in an object. In a transmission electron microscope (TEM), high voltage electron beam is focused by electromagnetic lenses and is illuminated on the specimen. Only a small part of transmitted electrons interact with the specimen and this then is magnified by lenses of an EM and projection of the magnified image of the specimen is recorded. Organic molecules and biological specimens like proteins are prone to radiation damage when exposed to electrons and a single exposure is sufficient to destroy them. Besides, biological specimens are composed of mainly light atoms and they scatter electrons weakly and images typically have low contrast. Thus, the amount of dose that can be used is a fine balance between visualizing them and preventing excess radiation damage.

Underlying all actions of our bodies are proteins that perform a wide variety of functions including transport, signal transduction, nerve conduction etc. Proteins made of amino acids, 20 of these occur naturally and in myriad of combinations they fold in three-dimensions (3D) and even a single change in one of these amino acids in certain proteins can cause disease, examples include sickle cell anemia, cystic-fibrosis. Thus, understanding how the amino acids in these proteins are spatially arranged in 3D and how they function and play important role in human health and in the design of drugs.

Traditionally, the techniques that have been used to obtain such 3D information of proteins include X-ray crystallography and Nuclear Magnetic Resonance spectroscopy. The former requires that protein of interest be coerced to form crystals and in later isotopic labeling is required. Sometimes, these processes can be laborious taking several years. As scientists, we would prefer to enlighten ourselves about such proteins by visualizing with a microscope to get an atomic model. Until recently, this was just a dream but recent technical advances in electron microscopy has made this possible and we can obtain atomic details of macromolecules just by imaging and averaging them as single molecules.
Thanks to the years of efforts by Jacques Dubochet, Joachim Frank and Richard Henderson and other scientists to the development of the electron cryomicroscopy, researchers like me can now explore the structure of bio-molecules without needing to make crystals to very high-resolution. Aptly, this year’s Nobel Prize for Chemistry has been awarded for this ‘resolution revolution’ which helped structure determination of biological molecules in solution. The award is timely as the field of structural biology has been revolutionized with these recent advances.

It is time to recall the steps that led to the development of the electron cryo-microscopy, which is much celebrated today.

  1. The introduction of low dose imaging led to structural determination of bacteriorhodopsin two-dimensional crystals by Richard Henderson and Nigel Unwin at Laboratory of Molecular Biology, Cambridge.
  2. It was realized that cooling the specimen to lower temperature for instance using liquid nitrogen to about -190˚C (instead of 23˚C) will reduce the radiation damage and higher resolution can be obtained. This was shown first by Ken Taylor and Bob Glaeser at UC Berkeley and subsequently, Jacques Dubochet and his colleagues developed the method of plunge freezing at EMBL, Heidelberg, which opened up the field of CryoEM.
  3. Though it was clear that macromolecules like proteins as single entities can be visualized in EM, due to signal to noise ratio, averaging of many similar molecules was necessary. The micrographs from EM are projections of an object and the aim of the single particle reconstruction is to relate these 2D projections in 3D. Joachim Frank and his colleagues developed the computational methods that allow us to perform the process of averaging and reconstruction.
  4. In 1995, Richard Henderson predicted that structure of molecules as small as 40 kDa can be obtained by single particle cryoEM with very little particles, provided some of the problems associated with EM such as radiation damage, detectors and beam-induced charging and movement are solved.
  5. The introduction of brighter and coherent electron source, better vacuum and stable stage in the microscope, faster and easier computing have made the use of EM as a biophysical technique more popular.

I still remember the tardy process of studying proteins in the ‘pre detector’ days of electron microscopy. As the life time of a given grid was short and we were imaging on films, only a limited number of films could be loaded and after exposure they had to be developed and fixed, rinsed with water, dried and then scanned. It was only at the end the entire day of hard work that we got to know whether the imaging was successful.

Thus, I was very excited when the new detectors were introduced few years back. The new detectors are based on CMOS (Complementary metal-oxide sensors) technology designed for electrons, which have higher signal to noise ratio and are called direct detectors. A useful analogy is to compare photography in 70-90’s to what it is now. From using film in the old days to store our memories, we have now moved on to digital cameras, which also use CMOS sensors.

The other major advantage of these new direct detectors is that they run in a movie mode. For comparison, let’s say we are watching the 100 m sprint in an Olympics. The finish is very close but we can look at the video, slow it down and figure out who crossed the line first. Similarly, we can now look at the sub-frames of a given exposure in an EM and study the effect of electrons on specimen and computationally correct for some of these effects.

This new detectors for EM has allowed scientists to determine structures of many important biological macromolecules that otherwise were very difficult such as the enzyme called gamma secretase that has been implicated in Alzheimer’s disease. Because, proteins that are difficult to crystallize can now be visualized and structure determined, screening and development of drugs is a possibility by EM. Another big advantage with cryoEM is the ability to trap different conformations of a macromolecule and computationally sort them, thus understanding the mechanism of a given protein.

I often tend to use Buzz Light year’s catchphrase in Pixar’s Toy Story ‘to infinity and beyond’ for cryoEM. It has a lot of potential and it is going to get even better in the next few years when few problems that are remaining are solved (better detectors and improved specimen preparation). It is already possible to visualize a number of proteins within the cell using electron cryotomography and this is one particular area where we will see more technical advances and will be a very popular technique not only for structural biologists but also for cell and neurobiologists.

India is also surging ahead in the path of using such state of the art facilities. At Bangalore Biocluster comprising of NCBS, InStem and C-CAMP, we have recently installed a high-end electron microscopy facility and I am hoping that I can share my experience and knowledge that I gained working with many imminent scientists including Richard Henderson with whom I was associated with until June of this year. This facility will allow Indian scientists to obtain 3D models of many biologically important molecules.

Vinoth Kumar Kutti Ragunath is a Faculty of the National Centre for Biological Sciences, Bangalore. He worked under Prof. Richard Henderson in the Laboratory of Molecular Biology as a post-doctoral fellow, where he sharpened his skills in structural biology including X-ray crystallography and latest developments in cryoEM.