Structural details of individual particles can be difficult to discern from raw images taken on an electron microscope. This is due to the low signal-to-noise ratio typical for images of radiation-sensitive biological samples. It becomes even more challenging when the particles are conformationally or compositionally heterogeneous.
2D class averaging is the first step in visualizing the three dimensional structure of a protein or macromolecule, and can provide researchers a comprehensive representation of particles in their sample. 2D class averages can help visually assess variability in qualities such as preferred orientation, conformation, aggregation, and homogeneity of a sample in order to help select the most viable samples for full 3D structure reconstruction.
2D class averages enhance signal to noise ratio, increasing information that can be gleaned from single particle analysis.
To obtain 2D class averages, first we vitrify grids by taking an aliquot of the sample in a buffer and applying it to a grid before plunging it into liquid ethane. The goal is to obtain a thin, uniform layer of ice, with particles embedded in a variety of orientations in order to capture a variety of different views of the sample. By imaging as many different orientations of the sample as possible, we are able to create a more accurate three dimensional reconstruction. 2D classifications can be obtained from both negative stain and cryo-EM micrographs.
In the 2D class averaging process, individual particles are selected from TEM images, aligned relative to each other, and then computationally classified based on apparent similarities. In this way each particle class becomes an average of many similar particles, which enhances the overall signal. These 2D class averages provide a rich source of information on basic particle features, domain structures, and can also help in understanding the degree of particle flexibility.
Our automated methods provide reliable high throughput image acquisition and processing, and help answer questions on critical particle features.
Structures We Can Perform 2D Classification On
Frequently Asked Questions
Can I see an example report showing Negative Stain or Cryo-EM 2D class averages?
When do you use negative stain imaging versus cryo-EM imaging for obtaining 2D classifications?
