Skeletal Muscle Research in Extraordinary Detail: A Case for Cryo-ET (Vlog)

Skeletal muscle research using cryo tomography

We recently had the incredible opportunity to go behind the scenes of the Department of Structural Biochemistry at the Max Planck Institute of Molecular Physiology. In this video series, we learn from Professor Stefan Raunser and his team about their lab’s expansion from single particle analysis with cryo-electron microscopy (cryo-EM) to cryo-electron tomography (cryo-ET) for skeletal muscle research. Together they share how this technique is dramatically increasing their ability to not only study ensembles of protein complexes but to better understand how they interact natively within a cell. They demonstrate such advancements through newly published muscle research, including the determination of a first-of-its-kind nebulin structure using cryo-ET.

This interview has been adapted for readability. For the full dialogue, please watch each video in the series.

What area of research are you focused on and how do you incorporate cryo-EM?

Raunser: My name is Stefan Raunser. I’m the Director of the Department of Structural Biochemistry at the Max Planck Institute of Molecular Physiology. We concentrate on muscle research and are especially interested in how single molecules modulate heart contraction which is then connected to heart disease.

In situ structure of nebulin determined with cryo-ET and skeletal muscle research

In situ structure of nebulin on the thin filament from mouse skeletal muscle resolved at 4.5 Angstroms. DOI: 10.1126/science.abn1934.

Cryo-EM is a major part of the Institute at Dortmund. In the last couple years, we switched part of our work from single particle cryo-EM to cryo-electron tomography (cryo-ET). The reason is that we got more interested in how the protein structures we solved are interacting with other proteins inside the cell in the natural environment.

We recently worked on psoas and cardiac muscle samples from mice. Using these tissues, we were able to solve the structure of nebulin that’s running along the thin filament of skeletal myofibrils. This is really fascinating because nebulin is a huge protein that is difficult to purify and no structure of nebulin had ever before been solved because of its large size. Now, we used cryo-ET to solve this muscle protein structure and elucidate its interaction with the thin filament, which is important to know for certain muscle diseases like nemaline myopathies.

Sebastian Tacke: I’m Sebastian Tacke, a post-doc at the Max Planck Institute of Dortmund responsible for hardware development and structural biology research. We are well equipped and have quite a lot of instruments from Thermo Fisher Scientific, including the Vitrobot to vitrify our samples, the Aquilos for cryo-FIB milling and the Hydra to mill thicker samples. We also have two Krios Cryo-TEMs.

Tacke: In the last three years, we have established a cryo-ET workflow. In the beginning, it took quite some effort to gain good data, including two years to get to a point where we had hundreds of good tomograms. Recently, a post-doc joined the group and within the first month, she was able to get the same amount of data but in a much shorter time. This, of course, makes all of us proud that we established this workflow and that it’s working so efficiently.

We hope that by demonstrating our improvements, it will help to accelerate the science itself because more people will realize that tomography is not a unique technique which can only be used in one or two labs worldwide. This is a technique which can be applied by scientists who are driven by biology and want to answer basic biological questions. The more people join this society, or this “tomo” group, the faster the science gets accelerated because more brainpower is put into it.

I’m quite sure that we’ll see quite some awesome research in the next five years related to tomography.

How do you envision the future of cryo tomography?

Tacke: In the future I imagine there’s a workflow where we can identify our region of interest with cryo-light microscopy, then we thin this region around our region of interest with cryo-FIB milling or plasma-FIB milling. With these techniques, we are able to take tomograms and get tons of lamella per day.

Wang: I’m Eric, a PhD student at Max Planck Institute of Molecular Physiology working with Professor Raunser. My main research focus is on the structures of sarcomere and the protein components within the sarcomere. I use electron cryo tomography to investigate protein structures in their native context. Cryo-ET will advance my research in the muscle field because it gives the power for us to investigate certain proteins that were not possible to be investigated previously with other techniques like single particle analysis or x-ray crystallography.

Raunser: Our dream in the end is that we are able to visualize how small molecules like drugs bind to specific proteins inside the cell. Electron tomography cannot only be used for our muscle research, but of course, you can imagine for cellular processes like septations or segregation of chromosomes or all kinds of different cellular aspects that really happen inside the cell and that you cannot reconstitute easily.

Cryo-electron tomography will be the key technique of the future.

This blog was written in collaboration with Stefan Raunser and Sebastian Tacke of the Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology in Dortmund, Germany.