Cryo-Electron Microscopy

Question 1

Biomolecules are studied in their _ state

Answer 1

Solution

Question 2

We will consider only _ not Scanning Electron Microscopy

Answer 2

Transmission Electron Microscopy (TEM)

Question 3

Brief overview of TEM

Answer 3

Similar to light microscopy, but sample is in a vacuum at cryogenic temperature and the lenses are electromagnetic

Question 4

Using electrons allows very high-_ images

Answer 4

Resolution

Question 5

Ideal sample size?

Answer 5

> 300kDa
Smaller samples have greater difficulties than larger ones

Question 6

Why must electron dose be kept low?

Answer 6

To reduce radiation damage

Question 7

Single-particle EM images are _

Answer 7

Projections

Question 8

What is a micrograph?

Answer 8

2D projections of 3D objects.

Question 9

True or false:
You only need one projection from each angle

Answer 9

False.
Images are noisy, so multiple are needed from each angle

Question 10

Basic idea of electron microscopy

Answer 10

Spread identical particles out on a film and image them.
Each particle is positioned at a different, unknown angle.
If there are enough images we can reconstruct the 3D shape of the particle

Question 11

Major advantage of Cryo-EM over X-ray Crystallography

Answer 11

Doesn’t require crystal formation.
Better for large complexes that won’t crystalise
Provides a more natural environment

Question 12

Major disadvantage of Cryo-EM compared to X-ray Crystallography

Answer 12

Typically lower resolution. Small proteins are difficult, because images of different angles look like very similar blobs.

Question 13

Negative staining

Answer 13

Particles are coated in heavy crystals to increase contrast.
But it limits resolution at can introduce artifacts
Sample is dehydrated which can lead to distortion/damage

Question 14

Vitrification

Answer 14

Particles are embedded in vitreous ice
Less contrast but allows higher resolution in a native-like state
Cryo-electron microscopy

Question 15

Why do negative staining or vitrification?

Answer 15

To allow the sample to survive in the electron microscope.
Usually stain first, then use vitreous ice to take high resolution images

Question 16

Radiation damage

Answer 16

Electrons are very damaging to biological molecules, so a low dose should be used
They can lead to very noisy images

Question 17

Why do particles move as they are imaged?

Answer 17

• Electrostatic effects of the beam
• Charge accumulation in vitreous ice
• Differential contraction of ice vs grid

Question 18

What does direct detection allow? (4)

Answer 18

• more accurate recording of image
• improved resolution
• greater sensitivity
• faster readout

Question 19

Cryo-EM workflow

Answer 19

• Data collection
• Particle extraction
• 2D classification
• 3D classification
• Refinement
• Modelling and Interpretation

Question 20

How to estimate relative orientation of particles?

Answer 20

By comparing with “simulated” projections from an initial model which is then refined further

Question 21

2D image analysis

Answer 21

• image pre-processing
• particle picking
• image clustering and class averaging

Methods to go from raw image data to higher resolution 2D projections

Question 22

3D reconstruction

Answer 22

Using the higher resolution projections to build a 3D model

• reconstruction with known view angles
• refinement with unknown view angles
• calculating initial structure
• fitting atomic-resolution models to lower-resolution EM structures

Question 23

What is motion correction?

Answer 23

Correcting for sample movement

Question 24

What is particle selection?

Answer 24

Finding the molecules in the images

Question 25

Sorting particles into classes based on orientation and structure, then align and average is a description of what?

Answer 25

2D classification

Question 26

Why are phases not lost

Answer 26

Electrons can be focussed using electromagnetic lenses.

Question 27

How many particles required for a typical reconstruction

Answer 27

50,000 Electrons interact more strongly than X-rays

Question 28

What are Thon rings?

Answer 28

Concentric light and dark rings in the power spectrum

Question 29

What does the Fourier Transform of the image depend on

Answer 29

Defocus, lens aberration and astigmatism

Question 30

Why can particle picking be difficult?

Answer 30

Because the images are low contrast and noisy and may also contain contaminants

Question 31

Particle picking methods

Answer 31

* manual picking * automated "blob" picking * automated template-based picking

Question 32

What is automated template-based picking?

Answer 32

Particle picking method relying on templates that come from: * 2D class averages of the other methods * low resolution projections of a known or predicted structure

Question 33

Averaging similar images reduces _

Answer 33

Noise It tends to cancel out

Question 34

What is the clustering problem?

Answer 34

We want to divide the projections into groups of similar view angles. We need a way to determine what those classes are and which images belong in them

Question 35

What is unsupervised learning?

Answer 35

Machine learning term for grouping images such that images within groups are similar and images in different groups are different

Question 36

What is 3D projection matching?

Answer 36

For each projection (i.e., each class average), find the view angle that best matches the 3D model Given the newly estimated view angles, reconstruct a better 3D model (e.g., using filtered back-projection)

Question 37

Combining tilted and untilted data gives _ results than just untilted.

Answer 37

Better

Question 38

Does symmetry help or hinder structure determination?

Answer 38

Help Need fewer particles. Fewer issues with preferential orientation

Question 39

What is Single Particle Analysis (SPA)?

Answer 39

* two-dimensional projections of randomly oriented particles * “purified” sample * particles in thin ice -> lower background -> higher resolution

Question 40

What is Sub-Tomogram Averaging (STA)?

Answer 40

* particles are represented by three dimensional volumes in a tomogram * sample in “native” environment * particles in a slab of frozen cellular material -> higher background -> lower resolution

Question 41

What is resolution in cryo-EM?

Answer 41

The particles are split into to sets and a map is calculated from each half The average resolution is the point at which the Fourier Shell Correlation between the two maps is below 0.143

Question 42

Density fitting is very dependent on _

Answer 42

Resolution There is difficulty of reaching higher resolution

Question 43

How to obtain atomic resolution models from lower-resolution EM?

Answer 43

Fit high resolution crystallography structures into the EM density.

Question 44

Validation of cryo-EM structures

Answer 44

Does the model agree with the map? Have we overfitted the data? Does the model look like other macromolecules and what we know of chemistry?

Question 45

Does the model agree with the map?

Answer 45

Global measure of how well the model fits the map Local measures of fit B-factor calculations

Question 46

Have we overfitted the data?

Answer 46

Cross-validation

Question 47

Does the model look like other macromolecules and what we know of chemistry?

Answer 47

Consistency of 3D structure with 1D sequence Deviations from ideal values (bonds, angles, etc) Non-bonded clashing atoms Stereochemistry (Ramachandran plot) Rotamers