Frozen in motion: capturing the open structure of channelrhodopsin

A curious gateway to science

Although both of my parents come from an art background, I felt immediately intrigued when I learned about ions in my junior high school chemistry class. However, I think I can say I was already interested in science since I was in elementary school, albeit in a different form. Reading detective novels was another gateway to science. In these novels, the killers often used poisons, and I was always surprised by their variety, which also got me interested in their mechanisms and ultimately how they interacted with the human body. That is where I feel the difference between chemistry and biology lies. Chemistry focuses more on the reaction and the target itself. Biologists, on the other hand, have an interest in the relationship between the target and what it is interacting with, be it DNA, cells, or the human body. I found investigating what is changing and moving rather than what is stable and motionless more captivating. So, I chose biology over chemistry. My decision solidified after entering college. When I was a first-year student, I took a structural biology class, which is now my major. In one lecture, the professor introduced the biological mechanisms of a certain protein, and I was enthralled. I wondered if in the future I would be able to uncover the mechanisms of a protein at the angstrom, one ten-billionth of a meter, level. However, only one year of research as an undergraduate proved to be too short. Luckily, my parents, my professor, and my lab mates all encouraged me to enter graduate school. I am glad to have taken the leap of faith and grateful for the support and care I have received along the way.

How does a cell door open?

Structural biology aims to determine the structure of biomaterials, such as proteins or DNA, and understand their mechanisms. Currently, I research channelrhodopsins, which, as the name suggests, are ion channels located in the cell membrane. One of their unique properties is that the channel opens when it is irradiated. In other words, when we shine light on the protein, its structure changes, and the ion can pass through from the side with a higher ion concentration. If I can determine the structure of channelrhodopsin at the angstrom level, I will be able to understand which part of the protein is responsible for the mechanism of this opening motion. This knowledge could then be leveraged to design more useful channelrhodopsins, which are already used in some research fields, such as neuroscience. However, there are still certain aspects that could be improved, such as energy transfer efficiency or the amount and specificity of ion transportation.

Catching channelrhodopsins in the act

I chose my target serendipitously: the professor giving the structural biology lectures that sparked my interest was working on channelrhodopsins. I have already determined some of their closed structures but gave myself an additional challenge: to determine their open structures. This is incredibly difficult as the timescale of channelrhodopsins is usually very fast. It takes about 50 microseconds (one millionth of a second) from the closed state to open. From there, they return to the closed state within 10 milliseconds (one-thousandth of a second) of irradiation. So, it is difficult to catch them in the act, so to speak. This is also the reason why most of the already determined structures of various types of channelrhodopsins are usually closed structures. I believe that clarifying the open state is more important for understanding which part is essential for its function. So, if I succeed, I will be able to contribute to the understanding of the functioning state of this type of channelrhodopsin.

Freeze motion, but for proteins

Luckily, most of the research procedure is already established. First, I prepare the DNA construct for the research target. In this day and age, there is abundant information online about various kinds of DNA constructs, and I can order the sequence I need on the internet. Then, I transfer the DNA construct to cells, such as human kidney cells, and use the cells as factories. Sometimes, when the cell struggles to produce the target protein, I cut and paste the DNA information to make it more suitable. Once the cells have produced the proteins, I purify and place them on a special grid. I usually use cryo-EM, cryo-electron microscopy, to collect the protein data. There are several of these machines on Hongo campus. I shine light on the target and freeze the grid to a very low temperature, under 100 Kelvin, to stop the movement of the protein. The hope is that I can capture the open structure using this method.

Separating the wheat from the chaff

The data I collect form a precise picture of the protein. The problem is that it is a mixture of signal, the actual protein, and noise, anything else the microscope picks up, such as ice or dust. I use software specifically developed to analyze such data. The goal is to get the correct density map, an image of the density in small clusters, by using software. This, however, is not possible by observing only one protein. I need to make several measurements to amplify the signal. Combining all of those signals results in the density map. One of the difficulties of my project, in particular, is that to determine the open structure of the protein, I cannot have a mixture of the closed and open states in my data. So, I do my best to extract the proteins such that I only select the open state.

Being on the UTokyo iGEM team in the middle of the pandemic

Actually, this is not the first time I have worked with irradiation. I was on the UTokyo iGEM (International Genetically Engineered Machine, an international synthetic biology competition) team as a first- and second-year student. I saw their fliers and decided to apply… and luckily passed the interview. Each iGEM project lasts a year, and in my sophomore year, our project involved irradiation and biosecurity. Our project aimed to create a unique code that could be revealed by shining light on the cell. A complication was that it was still in the middle of the pandemic, so the number of researchers allowed in the lab at the same time was limited. As I did not have that much research experience at the time, I had to start by reading many, many papers to catch-up. I also got to interview researchers and stakeholders in the biosecurity space. At the end of the year, the team went to Paris to present the project.

At the University of Sheffield

Last year, I also got to go to the UK as an exchange student. I chose the University of Sheffield, which is located in the middle of the country. I did not know anyone in my circle of friends who had gone there, so I was surprised to meet other Japanese exchange students. I even made friends with two UTokyo students. Although I was not allowed to conduct experiments as an exchange student, it was really refreshing to take lectures in biochemistry and biomedicine at a university famous for its biological research. Spending the entire semester there helped me appreciate the differences between teaching and learning styles in Japan and abroad. At UTokyo, lecturers, after introducing the basic concepts in biochemistry and biophysics, emphasize their research. They bring a lot of research papers to class to provide an overview of their field. At the University of Sheffield, lectures were more discussion-based, and the lecturers asked students many follow-up questions. It was a great opportunity to think about the implications of already established knowledge. I also took lectures on ethics, which I did not have time to do in Japan because I had lab practice every afternoon. It was a great opportunity to expand upon the knowledge I acquired as part of the mandatory research ethics class I took at UTokyo.

Do not hesitate to start something new

To be honest, I was afraid of going overseas. I took online English conversation courses, listened to BBC podcasts, and read papers written in English to prepare. However, the University of Sheffield is famous for accepting many international students, so support for international students was amazing. For example, I could rewatch a recording of the lectures with subtitles. Even when I could not understand what the lecturer said during the lecture, I could clarify the content right after class. I also got to meet so many kind people. Do not hesitate to try something new because many people will support you. I feel like even the not-so-great experiences enriched my life. So do not be afraid of trying something new.

※Year of Interview:2025

Photography:KAIZUKA Junichi
Text:Belta Emese
The interview was edited for brevity and clarity.

OBATA Hana

first-year master’s student in the Department of Biological Sciences

Hana Obata is currently a master’s student in the Department of Biological Sciences. Her research focuses on determining the open structure of certain types of channelrhodopsins. She was on UTokyo’s iGEM team as a first- and second-year undergraduate student. Her hobbies include reading, with a particular interest in mystery novels, and she has also developed an appreciation for science fiction since entering university.