[Repost] Cryo-EM: A chemical prize given to physicists, which has helped biologists
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Why Did Cryo-EM Win the 2017 Nobel Prize in Chemistry? By He Wanzhong (Researcher at the Beijing Institute of Life Sciences) In 2013, breakthroughs in cryo-EM technology brought about a revolution in the field of structural biology; it quickly swept through this field, enabling the determination of atomic-resolution structures of many important large complexes and membrane proteins that had long eluded solution using traditional X-ray and crystallographic methods. These structures were successively resolved, and their findings appeared frequently in top-tier journals and various media outlets. The atomic-resolution structures of important complexes determined by Dr. Cheng Yifan, Dr. Shi Yigong, Dr. Yang Maojun, and Dr. Liu Zhengfeng, in particular, shocked the world. This revolution in cryo-electron microscopy is characterized by its ability to rapidly resolve the three-dimensional atomic structure of large protein complexes, without the need for crystallization and with only a very small amount of sample required. There are two key technologies behind this breakthrough in electron microscopy resolution: direct electron cameras (in which Dr. Cheng Yifan and Dr. Li Xueming made significant contributions to the algorithms), and 3D reconstruction software. Behind these technological breakthroughs are three pioneers in the field of cryo-electron microscopy: Richard Henderson, Joachim Frank, and Jacques Dubochet, whose early and significant contributions were made in terms of fundamental theory, reconstruction algorithms, and experimentation respectively. I have had face-to-face interactions with all three of these scientists, and I also entered this field by reading their articles; below I will briefly discuss their contributions. The electron microscope was invented in 1931, but its application in biology lagged behind that in materials science. The reason is that biological samples must be moist to remain stable, whereas the electron microscope requires a high vacuum to function; thus, developing high-resolution biological samples for use in electron microscopes represents a technical challenge. The traditional heavy metal negative staining technique allows heavy metals to coat the protein surface, followed by dehydration and drying to produce samples suitable for vacuum imaging; however, this results in a reduced resolution of the samples (with a resolution of up to 1.5 nanometers). In 1968, Dr. Klug at the MRC Laboratory at the University of Cambridge in the UK and his student DeRosier developed a technique for the three-dimensional electron microscopy reconstruction of negatively stained phage viruses (Dr. Klug was awarded the Nobel Prize in Chemistry in 1982). But how can the atomic-resolution structure of biological samples be maintained while still being suitable for electron microscopy imaging? In 1974, Dr. Robert Glaeser from the University of California, Berkeley, and his student Ken Taylor first proposed and tested the use of electron microscopy for imaging frozen aqueous biological samples. This approach offered a new way to reduce the damage caused by radiation to high-resolution structures, as well as to maintain a high vacuum environment necessary for high-resolution imaging; this was the precursor to cryo-electron microscopy (CryoEM).
In 1982, a team led by Dr. Dubochet developed a truly mature and usable rapid freezing technique for preparing glassy ice-embedded samples that did not form ice crystals. With the development of cryostage technology, cryo-electron microscopy was officially introduced on a wider scale. Building on the three-dimensional reconstruction techniques proposed by Dr. Klug, Dr. Richard Henderson from the MRC Laboratory (with a background in physics and X-ray crystallography) and his colleague Dr. Unwin developed three-dimensional reconstruction techniques for two-dimensional electron crystals in 1975. This technique was then used to determine the three-dimensional structure of bacteriorhodopsin, a membrane protein involved in vision; the resolution achieved was 3.5 angstroms, which is an outstanding achievement. However, the first Nobel Prize awarded for the structure of a similar membrane protein went to the X-ray crystallographer Michel in 1988. The biggest problem with two-dimensional crystals is that it is difficult to grow them, which limits their range of applications; moreover, they are often overshadowed by X-ray crystallographers (I had my first project involving thin three-dimensional crystals taken away from me as soon as I started in this field). In the 1990s, Dr. Henderson turned to another emerging CryoEM 3D reconstruction technique, namely the single-particle analysis reconstruction method developed by Dr. Joachim Frank. This approach allows for the direct imaging of a series of protein or complex particles without the need for crystallization, followed by alignment, classification, and then 3D reconstruction. Leveraging his profound expertise in physics and electron microscopy, as well as his exceptional insight, Dr. Henderson proposed the feasibility of achieving atomic-resolution CryoEM technology. He made a series of forward-looking theoretical predictions, such as the need to address sample drift caused by electron beams in order to attain atomic resolution, which pointed the way for subsequent advancements in direct electron cameras; he himself was also involved in the development of such cameras. Therefore, in this revolution in electron microscope resolution, Dr. Henderson was a true pioneer. Furthermore, the breakthroughs in new 3D reconstruction algorithms were also attributed to Dr. Henderson’s keen insight; Dr. Sjors Scheres was recognized by him and recruited to MRC despite not having many published papers, and went on to achieve great success by developing the classic Relion 3D reconstruction algorithm. Finally, let us introduce Dr. Joachim Frank, who has developed the technique for three-dimensional reconstruction of single particles using cryo-electron microscopy; he also has a background in physics. Dr. Frank is the pioneer of single-particle analysis, as well as the inventor of the three-dimensional reconstruction algorithm for single particles and the software Spider. Frank studied under Dr. Hoppe, a renowned German electron microscopist. The Hoppe school advocated for the direct three-dimensional reconstruction of samples of any shape, and later techniques such as three-dimensional electron tomography and cryoEM three-dimensional reconstruction are all related to his early ideas. Dr. Frank proposed a method based on two-dimensional projection images of individual dispersed identical particles (proteins); by classifying, aligning, and averaging these images, the three-dimensional structure of the protein could be reconstructed. He developed a series of algorithms and created software (SPIDER) to enable the determination of protein three-dimensional structures without the need for crystallization. In particular, he carried out a series of important and groundbreaking works on the three-dimensional reconstruction of ribosomes; it’s a pity that he was not awarded the Nobel Prize for ribosome structure back then. It is well-deserved to award him a Nobel Prize for cryoEM single-particle 3D reconstruction now.
With this tool, progress has been made in other fields as well