Basic Concepts And Common Problems in Cryo-EM

Cryo-EM images are formed by pixels with different intensity in grayscale. Each pixel is represented as a number in the image file. Below is a table simulating the pixel intensity of an image. If we plot the numbers in the last row with column indexes, we may get a curve look like this: Here we need to introduce a theorem that every curve can be approximated by the sum of simple sine waves (y = A sin(wx +b)) as presented in the figure below. Therefore, if we apply Fourier transform to the curve, we will get a set of sine waves with different amplitude (A), phase (b) and frequency (1/w). In this way, we convert the information in the real image into three parameters in the Fourier space. And the process of converting a curve into several sine waves is called Fourier transform. The red curve on the left is able to be approximated by the sum of several blue curves on the right. Image from https://www.ritchievink.com/blog/2017/04/23/understanding-the-fourier-transform-by-exampl

1.        What is the preferred orientation problem? Preferred orientation problem occurs when most of the particles presented the same view in the EM image. It has been a tricky problem for high-resolution structure determination with cryo-EM, especially for samples without symmetry. It is caused by the adherence of the sample to the air-water interface or to the substrate of the grid (carbon film or graphene oxide). Let’s take 20S Proteasome as an example. Figure 1. The side view and top view (A) of the 20S proteasome (EMD-8726). The simulated image with the preferred side view and top view (B). When most of the proteasome particles prefer to “lie on the grid”, we will see a lot of side views like showing in Fig.1B (left panel). And when most of them “stand on the grid”, only top views are observed (Fig1B, right panel).  2.        How does the preferred orientation affect the resolution? To answer this question, we will need to first understand the “central slice