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Introduction of AMED/BINDS Cryo-electron Microscopy Facility at the University of Tokyo

Aims of Cryo-electron microscopy facility

Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) by Japan Agency for Medical Research and Development (AMED) aims to connect excellent life science research to the practical application of pharmaceutical products. Our facility was selected as one of the support platforms of the Structural Analysis Section in the structural analysis unit by AMED/BINDS in 2017. Because one cryo-electron microscope costs several hundred million yen, we manage cryo-electron microscopes as shared for structural analysis and develop and adopt new methods. The facility can provide about 7000 images or three terabytes of image data per day. The equipment utilization rate is 70% or more except during maintenance. The use of companies has been increasing recently.
In addition, from the fiscal year 2020, the University of Tokyo established the Collaborative Research Organization for Structural Life Sciences, which will focus on cross-scale structures including cells, tissues, and organs, forming a shared research center that can make full use of cutting-edge imaging methods.

What can we do with Cryo-electron microscopy?

In the 1990s, X-ray crystallography and NMR (nuclear magnetic resonance) have been mainly used to study molecular structures of proteins and nucleic acids. Such structural information is essential for understanding living organisms; however, since 2013, cryo-electron microscopy has been rapidly developing as another method for structural biology, thanks to technological innovation. This is because cryo-electron microscopy can analyze structures of membrane proteins and large complexes. Both membrane proteins and large complexes are difficult to be crystallized, making it difficult to use X-ray crystallography for those proteins.
　In contrast, the single-particle analysis of cryo-electron microscopy enables us to capture nanoscale structures of biomolecules in three dimensions from a small amount of the sample in a near-native state without crystallization. The value of this technology is evident from the fact that the 2017 Nobel Prize in Chemistry was awarded to its pioneers in cryo-EM.
To observe protein samples using cryo-EM, purified biological samples, which should be uniformly dispersed in solution, are quickly frozen in liquid ethane and kept frozen below -160 degrees Celsius. Samples are embedded in a thin amorphous ice, whose thickness is about 100 nm. The frozen samples are photographed with a transmission electron microscope. To obtain the three-dimensional structure of the target protein, the images of several hundred thousand molecules pointing in various directions are superimposed and averaged.
　This cryo-EM/single particle analysis is widely used to elucidate the structure of various biological samples. This was enabled by a combination of several techniques, such as the quick freezing method for samples, image analysis programs that reconstructs 3D structures from a large number of 2D images, direct electron detector (an ultrafast CMOS camera with direct electron beam detection), and the automated cryo-electron transmission microscope that can acquire a large amount of data. For example, the spike protein of the new coronavirus (SARS-CoV-2) and the structures of the G protein-coupled receptors, which are the target of many drug discoveries, have also been elucidated using this technique.

What can be done at the Cryo-electron microscopy facility in the University of Tokyo?

Among the cryo-electron microscopy techniques, the method mentioned in the previous section is called "single-particle analysis" and is used most commonly in our facility.
Tomography is another technique for analyzing irregular structures, such as the structures in cells. This is a method to construct a three-dimensional image by superimposing 2D images of a sample ≦300 nm in thickness which are shot with tilting range from -60 to +60 degrees in an electron microscope. Compared to the single-particle analysis described above, tomography has the advantage of directly observing relatively large and heterogenous structures, such as the cellular organelles. The structure of cilia that act as a cell's antenna or propeller has been elucidated by this method.
Another method, microED acquires electron diffraction images while rotating a small crystalline sample with a size of 100 nanometers or less to identify three-dimensional structures. Recently, the microED method is becoming more popular for the structural determination of organic molecules.

Cryo-electron microscopes available at our facility

(1) Titan Krios G4i

	Acceleration voltage	300kV
	Electronic direct detector	GATAN K3 BioQuantum Falcon 4EC
	Sample Stage	AutoGrid Inserted Side Entry Stage (Cryo Autoloader can transport up to 12 samples)
	Other equipment	phase plate
	Main Applications	Image Data Acquisition by High-Resolution Observation *Installed by support including "the program for promoting the enhancement of research universities" in the fiscal year 2020

(2) Titan Krios G3i

	Acceleration voltage	300kV
	Electronic direct detector	GATAN K3 BioQuantum, Falcon 3EC
	Sample Stage	AutoGrid Inserted Side Entry Stage (Cryo Autoloader can transport up to 12 samples)
	Other equipment	phase plate
	Main Applications	Image Data Acquisition by High-Resolution Observation

(3) Talos Arctica G2

	Acceleration voltage	200kV
	Electronic direct detector	GATAN K2 Summit, Falcon 3EC
	Sample Stage	AutoGrid Inserted Side Entry Stage (Up to 12 samples can be transported by Cryo Autoloader)
	Other equipment	phase plate
	Main Applications	Image Data Acquisition by High-Resolution Observation

(4) JEM-F200

	Acceleration voltage	80kV,200kV
	Resolution	0.27nm
	Electronic direct detector	GATAN K2 Summit
	Other equipment	phase plate
	Main Applications	Image Data Acquisition by High-Resolution Observation

(5) JEM-2010F

	Acceleration voltage	80k V,100kV,120kV,160kV,200kV
	Electron source	Thermal electric field emission type
	Resolution	0.155nm (lattice image)
	Main uses	Observation of stained and frozen samples

How to use this facility?

Please apply for the support consulting from BINDS one-stop window (https://www.supportbinds.jp/). In case we apply for this support, the "main unit requesting support" will be the "Structural Analysis Area" and "Research Representative" will be "(the University of Tokyo) Masahide Kikkawa (Electron Microscope)." Support will be started if it is judged that "support is possible" as a result of consulting.
The flow of the support will be as below:

1. Reserve the equipment you want to use through the person in charge
2. Prepare/send samples by the reserved date
3. Types of facility (Electron Microscope) use can be selected from technical support, exclusive use, trial use and so on
4. Transfer data if necessary
5. Confirm the usage fee, invoice the amount, and make payment by the due date

Conclusion

Until a few years ago, cryo-electron microscopy was available only for small and limited groups of people in Japan. By the AMED/BINDS support, many electron microscopes have been installed and can be used by researchers who are interested in structures. A cryo-electron microscope is not a magic tool that anyone can instantly solve structures. Sample preparation and data analysis sometimes require colossal effort. On the other hand, by using electron microscopy, several laboratories derived surprising results that rewrite textbooks, even though the lab were not familiar with conventional "structural biology" methods. If you have important questions that can be solved by "seeing" the structures of molecules and cells, please apply for the consultation.

Acknowledgment

I would like to thank Kazuhiko Nakamura and Toshie Furuya for assisting me in writing this column. I also thank the members of our lab for supporting this facility.

Masahide Kikkawa, M.D., Ph.D.
Professor, Graduate School of Medicine
The University of Tokyo