March 31 Flashcards
Instead of electron density, Cyro-EM map is called a ______ ____ map
Electric potential map
What resolution to see C-C bonds? What about protein secondary structure?
Need 1.5 A for C-C, and 3.5 A for beta sheets (a helic is 5.4 ish so 3.5 is good too)
What is used instead of physical lens to focus electron beam?
Electromagnets
Why want a thin sample (usually) for cryo- EM? Why would we want a slightly thicker sample?
Electrons don’t penetrate well, so need to blot excess liquid from EM grid so have thin sample. Thicker sample needed if protein needs lots of hydration around it
What is BIG diff between Cryo-EM and XRD data collection?
Cryo-EM does not lose phases when go into Fourier Space, but XRD loses phases
What is the image that we get from e beam hitting protein sample?
We get 2D projection of protein, hopefully protein is in diff orientations so we can sum all them to get image
Why can’t we use cryoprotectants for Cryo-EM samples?, What do we do instead?
They make it harder to blot away excess liquid, so sample is thicker and get larger noise:signal. We use liquid ethane to vitrify the sample instead
Proteins sit at air-ice interface, and may have preferential orientation. What can we add to limit this?
Detergent, it disrupts this interface
If use membrane mimetic for MB protein for Cryo-EM, is it going to be accurate to in vivo?
Not always
In Cryo-EM, what is shots per hole?
In Cryo-EM grid, have many holes. It takes a while to get microscope to focus on new hole, so try to take as many images of the hole as possible before moving on. Would move beam a bit each image as electron beam damages sample
Ideally, proteins would be orientated by a small angle, then if sum all these images we get good high res image. What is this ideal angle?
0.1 degrees
What are Cryo-EM Charging effects?
It is when damage caused to sample by electron beam
