Hello. I'm Grant Jensen, a professor of biology at the California Institute of Technology, and an investigator of The Howard Hughes Medical Institute. And welcome to this class, Getting started in cryo-EM. The class developed as a tutorial that I gave new members of the lab as they joined. And most of these individuals have been post-docs with strong backgrounds in biochemistry, structural biology. A lot of them had done quite a bit of X-ray crystallography. But in addition to them, there's also been several graduate students, undergraduates, and even a few high school students. And so the tutorial will aim at a broad range of potential students. We focus on basic principles of how the cryo-EM works. Now, there's a number of textbooks available that have been written for biologists on how to do biological EM. And I find that they don't have enough, you know, detail about image formation, and issues like the contrast transfer function and how it's corrected for the practicing structural biologist. On the other hand, there's a number of textbooks that have been written for material scientists and physicists. And they go into too much rigorous mathematical and physical detail. And so we have yet to find a textbook that's just right for an introduction to cryo-EM. And I'm also not aware of any good review papers that start at this very basic level of what are the elements of the electron microscope and how do they work. Thus, this class is intended to fill that gap, intended for anyone who wants to learn about cryo-EM. This course will prepare people for practical training on the microscope which is also essential to learn how to do cryo-EM. But the course will familiarize you with the vocabulary and the concepts that you'll need to understand before you can use it effectively. Learning to do cryo-EM is an iterative process. I've noticed that individuals in my lab have recorded this tutorial. And then I find them months later, even years later, I found them listening to it again. So by recording this as a video, I'm hoping that it will help. It will allow people to familiarize themselves with the concepts, then go to the microscope and practice what they've learned. But then come back to the videos, perhaps, to clarify issues that weren't as clear before. So we'll begin with an introduction about why use electrons. And that's just a few minutes. Then there will be seven parts in the whole series. The first part is called currents, coils, knobs and names. The basic anatomy of the electron microscope. There's two full hours of material here, divided into eight different modules. In this part, I'll talk about electron guns, electron lenses, the rest of the column, the sample chamber, energy filters, detectors, vacuum systems, and a little bit about safety. And the next part is called Fourier transforms and reciprocal space for the beginner. Again, this is about two hours of material separated into six different modules. And here, I'll explain what a Fourier transform is in one dimension, in two dimensions, in three dimensions. A little bit about convolution and cross-correlation. But I emphasize that this module really starts at the very beginning. The idea of sine waves being added. So if you're an expert and you already know all about Fourier transforms and reciprocal space, you might want to skip this part. Unless you think that in your future, you're going to be teaching this someday, and you'd like to see how I give it a shot. The next part is about image formation. Here, this is about three hours of material separated into six different modules. In this part, I'll talk about amplitude contrast and phase contrast, wave propagation and how adding waves with different phase shifts has different effects. And that'll prepare us to talk about the contrast transfer function, and defocus, and the effects of defocus. We'll talk about envelope functions, and finally, we'll talk about how to correct for the contrast transfer function, oscillations. Part four is fundamental challenges in biological EM. This will be one hour of material in five different modules. Now, the three main challenges are first, how do you preserve the native state biological material inside the ultra high vacuum of an electron microscope. Now, there's methods that are done at room temperature, and there's also methods involving freezing. We'll talk about all of those. We'll talk about different grids that are used as well. Then the second challenge is how do you obtain three-dimensional reconstructions from the projection images delivered by an electron microscope. The third fundamental challenge is radiation damage, so I'll talk about what we know about that subject as well. And the combination of these challenges and the possible solutions that have been invented so far lead us to the three basic modalities of cryo-EM. The first is tomography, and so part five is about tomography. Two hours of material and five different modules. The first module in tomography is just an introduction. Then we'll talk about the special issues of sample preparation for tomography specifically. Then we'll talk about data collection, how to choose all the different parameters that need to be chosen to record tilt series and produce tomograms. We'll talk about special issues of reconstruction involved in tomography. Then how to identify objects of interest inside the tomograms. And finally, the limitations of this technique. Part six is about the second basic modality in cryo-EM, which is single-particle analysis. This subject will take us three hours in six different modules. Again, we'll have an introduction to single particle analysis, issues of special concern, like how to create a good initial model. How to avoid reference bias, other variations on the basic work flow that's typically used, how to assess the resolution. Finally, we'll talk about the third basic modality in cryo-EM which is 2-D crystallography. Just an hour on this subject with four different modules. Again, an introduction, sample prep issues, particular points about data collection for 2-D crystallography and reconstruction. The limitations, and finally, just a note about helical tubes, how to image and process them. In total then, there's 14 hours of material broken into just over 40 different modules. The duration of the modules varies between just a few minutes, three or four minutes, to just over half an hour. So obviously, how you pace yourself is up to you. But hopefully, understanding the scope and the organization will help you as you make that decision. Now as you go through it; as you catch errors or you have comments or questions, I'd love to hear about them. Please send those to me at GettingStartedInCryoEM@gmail.com. I've established this account just for feedback on this class, and I'd love to hear from you.
