April 9

Question 1

In context of Cryo-ET, what is high structural density, visual overload, and lower signal:noise and denoising?

Answer 1

High structural density is as there are overlapping organelles and molecules, makes interpretatin challenging

Visual overload is when small regions contain dense clusters of objects, hard to disentangle structures

Low Signal:noise is that as biological samples are fragile, using lower electron doses causes less damage, but get lower signal. We can use donoising filters to enhance signal.

Question 2

What are pros/cons of manual and automated method of segmentation?

Answer 2

Manual can have high accuracy, but very time-consuming, and diff interpretations between people, not very scalable.

Automated can reduce time to do, but need training data, good quality of it, and generally computationally expensive, but can be done

Question 3

What is sub-tomogram averaging?

Answer 3

It is when have multiple copies of structures, so we get them independently, then align them to increase signal:noise, get higher resolution. We are merging 3D images.

Question 4

Sub-tomogram averaging is similar to single particle analysis, but the different conformations of any particles can be challenging as we get particles from their natural environments. What is a complication with dual alignment?

Answer 4

We need to align the raw tilt images. Also cells have lower number of particles to use than in purified sample. We therefore get lower signal.

Question 5

Why might we not see lysines, glutmate, aspartates missing from tomography?

Answer 5

Lysines too flexible, and electrons damage glutamate and aspartate by decarboxylating them.

Question 6

How use Cryo-ET to get order of protein complex assembly?

Answer 6

We knockout subunits 1 at a time, see what we get. Can get order, then get the individual segments and assemble them together to get full structureq