Lecture 5

Question 1

What is EM limited by ?

Answer 1

Sample preparation. The resolution of EM is limited by the resin we use

Question 2

Max resolution for EM?

Answer 2

50 angstrom

Question 3

Two ways to retrieve 3D images from 2D?

Answer 3

1. Serial sections
2. Tomography

Question 4

Serial sections

Answer 4

1. Cut the sample using the ultramicrotome and keep the sections in order(trapezoid)
2. Image each section in the EM
3. Stack of the sections to get the full 3D image

Question 5

How is serial sectioning different from classical EM?

Answer 5

Section the sections thinner

Question 6

Problem with serial sections

Answer 6

Resolution is Z is limited by the thickness of the section can only go up to about 30 nm

Question 7

What is tomography?

Answer 7

-Cut a thick section and put it in the EM and take pictures of it from various angles. We know the angle from which we took the image so then we construct the 3D image via back projection

Question 8

CT Scan

Answer 8

Human sized version of tomography

Question 9

Tilt series

Answer 9

When we collect the stack of images from various angles

Question 10

Problem with tomography?

Answer 10

-Can only deal with 300nm sections
-Cannot deal with a cell (too big)

Question 11

How do we deal with cells for tomography?

Answer 11

We do serial section tomography

Question 12

What is serial section tomography?

Answer 12

-Cut thick sections of various sections of the cell
Then do tomography on each section
Then stack all of the tomography sections together to get the full 3D image

Question 13

Problems with Fixation?

Answer 13

-Slow (glutaraldehyde is slow)
-Conformation changes of protein
-Permeability change of membreane
-Osmotic effect leads to dimensional alteration

Question 14

Problems with dehydration?

Answer 14

-Shrinkage
-Conformational change of proteins
-Loss of lipids

Question 15

Porblems with embedding?

Answer 15

-Shirnkage during polymerization
-Loss of lipids

Question 16

Problems with thin sectioning?

Answer 16

-Compression
-Knife marks

Question 17

Problems with staining?

Answer 17

-Artifacts

Question 18

Problems with TEM?

Answer 18

Interpretation mistakes

Question 19

How to overcome dehydration altering what we look at?

Answer 19

-Cryo-EM

Question 20

What is Cryo-EM?

Answer 20

-Frozen hydrated state

Question 21

Vitrification?

Answer 21

transformation of a substance into a solid state that is a non-crystalline amorphous solid

Question 22

How does Cryo-EM do vitrification?

Answer 22

By plunging a small volume of sample quickly into liquid Ethane at liquid nitrogen temperature
-Freezes the sample so fast that crystals don’t form

Question 23

Why does Cryo-EM imporve ultrastructure preservation?

Answer 23

-Instead of using fix we just freeze the sample which is faster
-No ice crystals form which would damage the sample
-Forzen hydrated( solid but in a liquid state(still hydrated))

Question 24

Leidenfrost Effect

Answer 24

The formation of a gas barrier between a hot surface and a boiling liquid if the temperature is great enough

Question 25

Why can't we vitrify the water in the sample using liquid nitrogen?

Answer 25

Temperature difference between the water in our sample and liquid nitrofen is so big the gas barrier will form and cause our sample to freeze slow which creates crystals

Question 26

Cryo-electron tomography?

Answer 26

1. Vitrify your sample 2. Put the sample in the EM and take tomographic pictures 3. Back reconstruction to get the 3D image

Question 27

How is cryo EM better than resin?

Answer 27

-Better ultrastructure preservation -Best resolution -Low contrast

Question 28

Why does cryo-EM have low contrast?

Answer 28

-Limit amount of electrons we can use on the sample since it is still hydrated

Question 29

What are the benefits of visualizing atomic details in Cryo-EM?

Answer 29

Understansing things at the molecular level(how ribosomes do translation)

Question 30

What technique allows us to visualize atomic details?

Answer 30

Single particle cryo-EM

Question 31

Requirement for single particle EM?

Answer 31

Must use purified protein complexes not organelles or cells

Question 32

Particle Picking?

Answer 32

Choosing the particles in the cell that are at varying orientations

Question 33

2D classification

Answer 33

Grouping of the ribosomes based on their appearance, same orientation, same conformation or same composition

Question 34

3D alignment & reconstruction

Answer 34

1. Match our expierimental image with the 3D projection(whichever one matches best) 3. Then do tomography and do backreconstruction to get the 3D structure

Question 35

Cryo-EM for structure determination advantages?

Answer 35

-Easy sample prep -Molecules close to native state -Requires only a small amount of sample -More forgiving on sample purity

Question 36

Jacques Dubocht

Answer 36

Vitrification

Question 37

Joachim Frank

Answer 37

Computational of the 3D structures

Question 38

Richard Henderson

Answer 38

Instrumentation and Imaging