Cheng Xing talks about the development of cryo-electron microscopy

If two years ago, we said cryo-EM is an important tool for structural biology research, many people should disapprove. After all, although cryo-electron microscopy and X-ray crystallography and nuclear magnetic resonance have been called the three major tools of structural biology research, they have to admit that cryo-electron microscopy is the weakest of the three technologies. It has now resolved more than a thousand Among the membrane protein structures, more than 90% use the X-ray crystallography method, and NMR plays an important role in the analysis of the protein structure of small molecular weight.

However, on December 5, 2013, Associate Professor Cheng Yifan of the University of California, San Francisco, and colleagues David Julius collaborated on the use of a single-electron counting detector with near-atomic resolution (3.4 angstroms) to determine that in pain and heat perception The exciting effect of the central role of a membrane protein, TRPV1, has enabled researchers to revisit the role of cryo-electron microscopy in the study of structural biology. After all, compared with the X-ray crystallography method, it requires a small amount of sample and does not need to generate crystals, which brings new hope for the study of some hard-to-crystallize proteins.

Recently, during the “2014 International Seminar on the Freezing Electron Microscope Three-Dimensional Molecular Imaging”, instrument information network editors specially interviewed Cheng Yifan who came to attend the conference. He asked him to introduce the impact of the new detectors used in the research on the resolution of cryogenic electron microscopy. Does the development of cryo-electron microscopy mean the end of the era of X-ray crystallography? The future direction of cryo-electron microscopy and the issues that need attention?

Cheng Yifan, an associate professor at the University of California, San Francisco, used direct electron detection technology to achieve a near-atomic-resolution resolution of the cryo-electron microscope. Although people have long realized that cryo-electron microscopes have the potential to achieve atomic-level resolution, it is still necessary to achieve this. Many challenges. In the research of Cheng Yifan, his laboratory participated in the improvement of the cameras used in cryo-electron microscopy and the development of single-electron counting detectors. A single-electron counting detector is a direct electron detector that can directly detect electrons without converting electrons into photons as in conventional CCD cameras, and then converting them into optoelectronics for detection.

Cheng Yifan introduced: “The traditional DQE (detection quantum efficiency) of traditional CCD cameras is only about 30% at low frequencies and is even lower at high frequencies, which seriously affects the collection of high-resolution information. Therefore, new detectors have been developed to increase the resolution. It is an important subject for the study of cryogenic electron microscopy, which was mainly promoted by my colleagues and collaborators David Agard at UCSF, Professor Richard Henderson at MRC in the United Kingdom, and several professors at UCSD. It is foreseeable that camera development is an important issue for electron microscopy. It can be said that David Agard was very far-sighted. He foresaw the importance of single-electron counting and decided to work with Gatan as a single-electron counting detector. Several companies find it difficult to have practical applications for single-electron counting."

“Beginning in 2010, we have worked with Gatan and the Lawrence Berkeley National Laboratory for more than three years, and we continue to explore without seeing any prospects. Eventually we read quickly and almost noiselessly. Counting combination, the camera's detection quantum efficiency was raised to 70%.We also found the best conditions for using a single-electron counting camera through experiments, combined with the improvement of the software algorithm, the image blur can be corrected, and the near-atomic resolution is achieved. "Cheng Yifan said.

Currently, this single-electron counting camera has been commercialized by Gatan, the K2 Summit Direct Detection Camera. The other two companies (FEI and Direct Electron) also introduced direct electronic detection cameras, but none of them are single-electron counting. According to reports, direct electronic probing cameras are relatively expensive and are a large investment for any unit. If they are to be promoted, prices will be a key limiting factor. “But now it seems that there are no direct electronic detection cameras that almost determine whether there is a chance for an electron microscopy laboratory to have a place in the fierce competition. It's like deciding in a game that you are a player on the court and still present. The audience outside. This has exerted great pressure on those electron microscope laboratories that have not equipped such kind of camera now. " Cheng Yifan says.

Does the development of cryo-electron microscopy mean that the X-ray crystallography era is coming to an end?

It is tantamount to a revolution without directly crystallizing the protein structure and reaching near-atomic resolution. So, does the development of cryo-electron microscopy mean that the X-ray crystallography era is coming to an end? In the view of Cheng Yifan, it will not be, and it will not be in the future.

He said: “For now, although the resolution of cryo-electron microscope has been greatly improved, for example, we can see the binding sites between small molecules and proteins, but we can't see clearly how small molecules and proteins are combined. At this point, there is still a gap between cryogenic electron microscopy and X-ray crystallography, and now the development of cryo-electron microscopy technology has attracted the interest of people doing basic research, but pharmaceutical companies are still watching. The rate can reach about 2 angstroms, which is very helpful for drug design and screening, and pharmaceutical companies invest much more in R&D than basic research, and if they approve of this technology, it will bring a big market. Now apply X In the laboratory of the ray crystallography technology, electron microscopy technology will be used in the future, and in this case, many research institutes and pharmaceutical companies will not only deploy one electron microscope, but several electron microscopes, and their demand for cryogenic electron microscopy will also be corresponding. It will increase.Compared with X-ray crystallography, there are too few students in the laboratory of cryo-electron microscopy to meet the demand. ”

“In the future, it is not enough for a laboratory to master only the same technology. In the structural analysis, we can do it in different ways. Without a fixed model, we can see which way is faster. In addition, the future structural organisms. The study of science is no longer merely an analytical structure, but focuses more on solving practical biological problems.The systematicness and complexity of biological problems need us to solve from various aspects and combine various technologies, and a single technology is certainly not enough. Therefore, with the development of the cryo-electron microscopy technology, it will become a complementary and complementary technical method to X-ray crystallography in structural biology research, instead of replacing it, said Cheng Yifan.

The development of cryo-electron microscope still need to solve and pay attention to what problems?

Regarding the problems that need to be solved in the development of cryogenic electron microscopy, Cheng Yifan stated that the main objective is to further increase the resolution and to form a streamlined operation.

Cheng Yifan said: "Although compared with the past, the resolution of the cryo-electron microscope has greatly improved, but it is still hovering around 3 angstroms, as if there is an invisible wall. If it can further break through to 2 angstroms, it will be a This is a very challenging and exciting thing, but we still do not know what the specific reason is that it has limited the improvement of resolution. Improvements in instruments, cameras, sample preparation, software, etc. may bring new breakthroughs."

In order to form a process operation, Cheng Yifan said: “At present, electron microscopy has not yet formed a streamlined operation like X-ray crystallography. It is difficult for a laboratory to master this technology within 1-2 years. We hope that in the future 3 - Within five years, the technology of cryogenic electron microscopy can be streamlined, and the technical barriers to entry in this field can be reduced, so that more people can accept and understand, so that the cryo-electron microscopy technology can be truly promoted."

During the interview, Cheng Yifan raised his own concerns and opinions on the cultural atmosphere of the cryogenic electron microscope research circle. He said: "The development of technology has great impact on culture, and sometimes we may not realize it. At present, the culture of X-ray crystallography and cryo-electron microscopy are very different. X-ray crystallography is relatively mature. Therefore, the research period of each sample is relatively short, competition is fierce, and there is little communication between each other, and their respective research contents are also kept confidential before publication. In the field of cryo-electron microscopy, in the past ten years, we They all know what each laboratory is researching, and then try to avoid doing the same thing.The first one is because the fields are small and everyone is more friendly; the other is that the research cycle of the cryogenic electron microscope is relatively long. It has already begun to do, and later people want to exceed it."

"But now, with the development of cryo-electron microscopy, the entire research cycle has gradually become shorter and shorter. If one person is doing it, another person who sees improvement after doing it is likely to produce results first, which also leads to electron microscopy. People in the field are becoming more and more careful and confidential, and confidentiality is necessary in some aspects, but it will also become an obstacle to the development of technology. Facing this kind of realistic trend, how do we adapt to it without losing our inherent excellence? Cultural tradition, this is indeed a challenge."

Finally, when asked about research topics to follow, Cheng Yifan stated: "My own interest is still the study of membrane proteins or ion channels. In addition, I also hope to do something challenging. Others may feel that they can't do it. Research topics."

In addition, Cheng Yifan has also been particularly interested in the electron diffraction technology of cryo-electron microscopy, and believes that people engaged in X-ray crystallography must pay attention to this technology. He said: “One of the problems often encountered in the study of X-ray crystallography is that the crystals are small and can only form very small crystals. The current solution to this problem is to use X-ray free electron lasers, which are currently This type of light source is only available in Japan, the United States, and Europe, and the cost of light source construction and use is very expensive. Now we can use cryogenic electron microscopy to fully analyze the structure using very small crystals. This is freezing. A completely new application area of ​​electron microscopy technology is very worthy of attention.”