Lecture 5 - Electron Microscope 2

Question 1

retrieving 3D information from 2D images

Answer 1

serial sections: technique that involves cutting resin embedded bioloigcal tissue into hundreds or thousands of 30-50 nm thick, consecutive slices, which are then imaged using TEM to reconstruct structures.

tomography: 3D imaging technique in EM that reconstructs the internal structure of samples at 2-20nm resolution by assembling series of 2D images taken at different angles.

Question 2

serial sections

Answer 2

Serial section from sectioning allows 3D view of the
structure. Resolution is limited by section thickness
stack up images.

new vision of the ER
- stacked ER sheets are connected by helicoidal membrane motifs.

Question 3

Tomography

Answer 3

allows to get better 3D information about structures in the thickness of the sections
limitations:
- Technically demanding
- Requires thicker sections
- Time-consuming
- Radiation damage possible

Question 4

Potential Problems with conventional EM samples preparation

Answer 4

fixation: slow, conformation changes of protein, permeability chnage of membranes, osmotic effect leads to dimensional alteration, loss of diffusible ions and small molecules, masking of antigens
dehydration: shrinkage, conformation change of proteins, loss of lipids
embedding: mechanical effects, shrinkage during polymerisation, loss of lipids
thin sectionning: compression, knife marks
staining: staining artefacts of precipitation of heavy metals
TEM : interpretation mistakes

Question 5

Vitrifying water

Answer 5

Vitrification is the transformation of a substance into a glass that is to say a non-crystalline amorphous solid
Cyo-EM is the vitrification of biological samples in solution on the EM grid by plunging a small volume smaple quickly into liquid ethane at liquid nitrogen temp.

Question 6

Why do cryo-EM improve ultrastructure
preservation?

Answer 6

instead of using chemical to fix smaple, we could freeze it really fast
ice crystal are damaging
if we freeze very fast, ice crystals don’t forms
the sample is frozen hydrated ans in its native state.
Leidenfrost effect

Question 7

Why Liquid Ethane, Not Liquid Nitrogen

Answer 7

Leidenfrost effect: formation of a gas barrier between a hot surface and a boiling liquid if the temperature difference is great enough

Vitrifying water with liquid nitrogen will not work because
of the Leidenfrost effect
* Liquid Nitrogen boiling point -195.8 °C
* Liquid Ethane boiling point -89 °C
* Water at room temperature 20 °C

Question 8

can we see proteins directly in the cell in cryo-EM?

Answer 8

thin cell area or cellular organelles -> cry-EM tomography
tomogrpahy structures of nCoV spikes

Question 9

Cryo EM vs Resin

Answer 9

cryo : best ultrastructure preservation, best resolution, but low contrast image
resin: poor structure preservation, low resolution but best contrast

Question 10

Single particle analysis

Answer 10

Thousands to millions of images of identical protein particles are combined to reconstruct a high-resolution 3D structure
requires purified protein complexes, not organelles or cells.
1. freeze proteins, which are then applied to a grid
2. in the microscope, image what you see
3. classify images and assign orientation to get the 3D structure.

Question 11

can we see atomic details in cryo-EM?

Answer 11

higher resolution:
- understand how the cell functions at the molecular level (protein level)
- to understand how proteins function
- single particle EM
in resin section, corona viruses are seen as black ball.

Question 12

SPA

Answer 12

particle picking: Identify the location of the protein complexes (particles) in the image
2D classification: 2D images need to be grouped based on their appearanc
3D alignment & Reconstruction
3D Classification reveals conformation dynamics: resolve conformational dynamics

Question 13

sample prep for cryo em

Answer 13

purify the sample, choose and prepare your grid (holey carbon on Cu/Au), apply sample to grid, blot to make a thing film, vitrify, store and load for imaging.

Question 14

advantages of Cryo-em

Answer 14

easy sample prep
molecules in close-to-native state
requires only mall amout of sample
more forgiven on sample purity
give information on smaple dynamics

Question 15

structure of 2019-nCoV spike in prefusion conformation

Answer 15

3D variability analysis
understanding coronavirus variant:
- N501Y mutation on one of the 3 spike proteins, and cannot attach to human receptor?

Question 16

what can we see using single particle cryo-em

Answer 16

ribosome
microtubules
actin filaments
tau filament from brain of patient