Negative Stain for High Resolution Cryo-EM Workflows

You can see a lot just by looking...

As Yogi Berra did or did not say: "you can see a lot by just looking". That is why the first thing we usually do when our clients send us protein samples planned for high resolution protein structure determination, is take a first "look" in negative stain. Negative stain analysis is a method that uses a heavy metal salt solution to lightly coat biological samples. This coating increases image contrast and allows sensitive samples to better withstand the high vacuum and high radiation environment of the transmission electron microscope (TEM). We use the images to evaluate if the sample:

• consists of uniform, monodisperse particles
• is the expected size and approximate shape
• is the expected concentration
• is falling apart or forming aggregates.

We can usually do this evaluation by just taking a handful of images of the sample. But if we want to see more, we need to look harder. For example, we might want to understand the domain structure of a small membrane protein, or the flexibility of an antibody, or whether a protein is forming the expected complex.  To achieve this deeper look, we first optimize our negative stain protocol, making sure we have stain that is not too thick and not too thin, and that the particles are nicely distributed, neither overlapping nor too sparse. Then we acquire many images (10’s-100’s of thousands) of these optimally stained particles so that we have 1000’s of examples of our particles of interest. Next, we carefully select individual particles, cut them out of the overall image, align them to each other and group together those that are of a similar shape and in the same orientation, a process known as 2D classification.  The 2D classes are an average of many individual particles and have a greatly improved signal to noise ratio. This allows us to see details of the domain structure of the protein or protein complex. We recommend this careful examination of protein samples in advance of undertaking sample preparation for cryo-EM for most samples destined for our single particle analysis workflow.

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