Imaging molecules

Jacques Dubochet of Switzerland, Richard Henderson of the U.K. and Joachim Frank of the U.S. have been awarded this year’s Nobel Prize in Chemistry for their contribution to cryo-electron microscopy, which enables high-resolution imaging of protein molecules.

Richard Henderson, born in 1945 in Edinburgh, Scotland. PhD in 1969, Cambridge University, U.K. Programme Leader, MRC Laboratory of Molecular Biology, Cambridge, U.K.

A REMARKABLE visualisation technique that makes possible three-dimensional (3D) capture of biomolecules in atomic detail has won this year’s Nobel Prize in Chemistry. The prize was shared equally by three scientists—Jacques Dubochet from Switzerland, Richard Henderson from the United Kingdom and Joachim Frank from the United States—for their pioneering work that led to the development of cryo-electron microscopy (cryo-EM), which brought to light the chemistry of life in amazing detail.

“Using cryo-electron microscopy, researchers can now freeze biomolecules mid-movement and portray them at atomic resolution. This technology has taken biochemistry into a new era,” the Nobel Prize Committee said.

All three scientists, working mostly independent of one another, focussed on different aspects of electron microscopy, a technique that has been in existence for over eight decades. But it was thought to be suitable only for studying dead matter because when a powerful beam of electrons—which is used in electron microscopy as against light used in light microscopy—passes through a sample, it could burn through the biological material, defeating the whole purpose.

The prize-winning laureates’ discoveries over a span of four decades helped optimise “every nut and bolt” of the electron microscope, helping it re-emerge as an indispensable tool to study the molecules of life.

While Dubochet, now an honorary professor of biophysics at the University of Lausanne in Switzerland, developed a technique to flash-cool biomolecules in a thin layer of water or other solutions, Frank, a biophysicist at Columbia University in the U.S., devised methods to turn thousands of serially taken images into a finely detailed 3D representation. Henderson, who works at the MRC Laboratory of Molecular Biology (LMB) in U.K. (where the 2009 Nobel laureate for chemistry, Venkatraman Ramakrishnan, also works), was the first scientist to create a 3D snapshot of a protein at atomic resolution using an electron microscope in 1990.

“Their work has paved the way for the recent advances in electron microscopy that are revolutionising structural biology by making it possible to determine atomic structures of large macromolecules without the use of crystals,” said Ramakrishnan, who is also the current president of the Royal Society.

Structural biologists have been using the cryo-EM technology to unravel the numerous structures of life’s molecular machinery. They include microbes attacking cells, proteins that cause resistance to antibiotics and other drugs, and complex chemical reactions involved in the process photosynthesis. Recently, scientists in Brazil studying the Zika virus, which is suspected to trigger brain damage in newborns, turned to cryo-EM to create high-resolution 3D images of the virus. Understanding the structure of the virus could help researchers identify potential targets in the virus to block its activity. This would, in turn, pave the way for developing drugs and vaccines that can help contain the epidemic.

Evolution of techniques

For a good part of the 20th century, scientists did not have a tool to image biomolecules such as proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) even though they knew that these complex molecules played fundamental roles in the cell. This changed in the 1950s, when scientists began exposing protein crystals to X-ray beams, enabling them to visualise these biomolecules’ waves and spiralling structures. About three decades later, nuclear magnetic resonance (NMR) spectroscopy, a technique capable of studying proteins in solid form as well as in solutions, came on the scene. NMR spectroscopy had other advantages as well. In addition to revealing protein structures, it could also show how these proteins moved and interacted with other molecules. But, both these techniques suffered from certain fundamental limitations. While X-ray crystallography could only study molecules that formed well-organised crystals, NMR spectroscopy’s scope was often limited to relatively small molecules.

Henderson, who, in his early academic career, had used X-ray crystallography at the LMB, abandoned it in the 1970s as many molecules failed to arrange themselves in crystals. He began exploring the idea of using electron microscopy as an alternative.

“In the field of material science, electron microscopy is often used to achieve resolutions of one angstrom or better (one angstrom, or Å, is equal to one-tenth of a nanometre). However, unlike these inorganic materials (such as steel or other metals), organic molecules or biological specimens are prone to radiation damage when exposed to electrons,” said Vinothkumar K.R., a scientist at the National Centre for Biological Sciences (NCBS), Bengaluru, who once worked in Henderson’s laboratory as a postdoctoral researcher. A single exposure to powerful electron beams was enough to destroy the protein, he said. Vinothkumar joined Henderson in 2006. Although they worked together in the initial years, Vinothkumar later pursued his research independently until he left the LMB in June this year.

Between 1970 and 1975, Henderson, along with his colleague Nigel Unwin, created a 7 Å map of a membrane protein called bacteriorhodopsin, a purple-coloured protein that is embedded in the membrane of photosynthesising organisms. This was groundbreaking work in both electron microscopy and in the study of membrane proteins. Subsequently, other research groups established that lowering the temperature of the specimen further prolonged its life, increasing the chances of obtaining better resolution, said Vinothkumar.

Henderson’s way of using electron microscopy still suffered from a serious drawback. It did not work for water-soluble biomolecules. Many researchers tried freezing the samples before the procedure as ice evaporates more slowly than water. But that too did not work as the ice crystals disrupted the electron beams, resulting in hazy images.

“This led to the work of Dubochet, who was then at the European Molecular Biology Laboratory (EMBL) in Heidelberg. He developed the method of plunge-freezing, thus opening up the field of cryo-EM,” Vinothkumar said. Dubochet found that if water was cooled rapidly, it solidified in its liquid form to form glass, also known as vitrified water, instead of crystals. Dubochet realised that if he could get water to form glass, electron beams would diffract evenly and provide a uniform background. And this actually worked.

Then came Frank’s work, which made the technology widely applicable. “Between 1975 and 1986, Frank developed an imag-processing method in which the electron microscope’s fuzzy two-dimensional images are analysed and merged to reveal a sharp three-dimensional structure,” the Nobel Committee said.

According to Vinothkumar, a useful analogy to understand the revolutionary changes in electron microscopy would be to compare photography in the 1970s, the 1980s and the 1990s to what it is now. “In the old days of photography, a person with a camera had to load a roll of film; after taking a picture, this had to be developed in a lab and printed. If there was a problem during exposure (for example, people moving or settings being incorrect), one would come to know about the quality of the picture only after it was printed. Today, we have digital cameras, which also use CMOS sensors and the picture taken can be seen immediately and any mistake can be corrected or a retake can be done immediately,” he said.

The cryo-EM technology took a while to get established in India. This was primarily due to the cost associated with the equipment and its running costs. In recent years, there has been progress in this area with the purchase of instruments and the hiring of experts needed to poerate them. Although there are a few high-end instruments for material science, there are only two for life sciences research. The first one was installed at the Indian Institute of Chemical Biology in Kolkata; the second one, procured recently by the Bangalore Biocluster—comprising the NCBS, the Institute for Stem Cell Biology and Regenerative Medicine (InStem) and the Centre for Cellular and Molecular Platforms (C-CAMP)—is equipped with all the necessary tools for high-resolution structure determination, Vinothkumar said.