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Protein Structure & Function > Imaging Techniques > Flashcards

Imaging Techniques Flashcards

1
Q

Why is it important to understand a protein’s 3D structure?

A

Proteins function is dictated by 3D structure = catalytic residues in active site and how proteins interact with other proteins

Allows us to create hypotheses about how to affect/control/modify it = change its function or predict molecules that bind to that protein

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2
Q

What resolution does single-particle cryo-EM look at?

A

Structural biology, near atomic resolutions so can see side chains and proteins

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3
Q

What resolution does cryo-electron tomography look at?

A

Proteins > macromolecular machines>bacteria

This is the gap between structural and cell biology

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4
Q

What are the uses of cryo-ET in infectious disease?***

A

Visualise pathogen structure and assembly

Can understand how the pathogen interacts with its target

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5
Q

What do cryo-ET subtomogram averaging and single particle cryo-EM look at?

A

Cryo-EM = whole cells, unique and heterogeneous

Single particle = purified macromolecules, isolated and homogeneous

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6
Q

What is the difference between resolutions for cryoET/EM?

A

Cryo-ET looks at the native and complex, resolution increases

Single-patricle = looks in vitro with high resolution

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7
Q

What is important in biological sample preparation?

A

Preserve the natural integrity
Thin layer of suspension

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8
Q

How is the natural integrity of the sample kept?

A

Hydrated at low temperatures and in a vacuum = no crystal ice forms and it is not dry

Inactivation for biosafety***

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9
Q

What is cryofixation?

A

Sample preparation method

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10
Q

How can they use such low temperatures but not have ice?***

A

Liquid ethane heat transfer much quicker
Water molecules don’t have time to crystallize

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11
Q

Explain manual vs automatic plunger for freezing?

A

Manual uses a steel rod to plunge the sample in quickly

Automatic plunger uses vitrobot

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12
Q

What are the different methods of cellular sample preparation?

A

Room-temp EM = high pressure freezing for thickness <200nm

Cryo-EM = plunge freezing for thickness <20nm

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13
Q

What happens when sample is thick?

A

Higher kV is needed

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14
Q

How does high pressure freezing (HPF) work?

A

Increased pressure means temp for freezing is reduced

At pressure 2045 bar = freezing temp -92

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15
Q

What is cryo-FIB milling?***

A

Cryo-focused ion beam microscopes are used to thin a sample

The focused ion beam is used to prepare a thin, electron-transparent lamella by removing material above and below the target region

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16
Q

Why is the sample coated in metal for cryo-FIB?

A

To increase electron conductivity

17
Q

What is a polyQ inclusion?

A

A polyQ inclusion refers to abnormal aggregates of proteins containing extended polyglutamine (polyQ) repeats.

Polyglutamine is a sequence of glutamine (Q) residues, and when present in excessive lengths, it can cause the protein to misfold and form aggregates.

18
Q

What do we need to think about once the sample is ready to go?

A

Which microscope to use?
Which parameters to set up?
How to collect data?

19
Q

How do you get 3D structure from TEM?

A

Electrons pass through the biological sample and the camera below records only 2D projections

20
Q

How do you get 2D projection from different views?

A

Single particle cryo-EM = different views in one single micrograph are imaged from many copies of the homogeneous sample orientated differently

Cryo-ET = different view in different micrographs are imaged from one unique sample which, which is physically rotated on the microscope stage

21
Q

What machine is used for high-resolution cryo-ET and subtomogram averaging?

A

Titan Krios = 300kV can penetrate thick samples
Direct Electron Detector(DED) w energy filter
Optimized stability to reduce drift

22
Q

How are the samples on cryo-TEM screened?

A

Look at the grid map
Select region of interest in the grid squares
Zoom in to check the ice thickness = med magnification
Target the features for data collection = high magnification

23
Q

What needs to be taken into account with tilt series?

A

Tilt range = completeness of information and sample thickness
Electron dose = signal-to-noise ratio and radiation damage
Angular increment = dose and signal distribution

24
Q

What are the units for electron dose of sample?

A

e- / A^2

25
Q

What is the tilt series scheme they established was most effective?

A

Dose-symmetric tilt series scheme
From -60 to +60 in 3 degree increments

26
Q

What are the two ways of projection?

A

Forward projection = imaging
Back projection = reconstruction

27
Q

What is subtomogram averaging?

A

Collection of subtomograms from one tomogram to give better resolution

28
Q

How does subtomogram averaging work?

A

Particle picking
Lattice map
Subtomogram extraction and alignment

29
Q

What are the steps for subtomogram extraction and alignment?

A
  1. Subtomograms = extracted from reconstructed tomograms
  2. Rotational and translational alignments are performed against a reference
  3. Aligned subtomograms are averaged to generate a new reference
  4. The new reference is used for iterative alignments
    Procedures 1-4 are repeated until the reference stabilizes
30
Q

How do they reconstruct a 3D structure from 2D projections or 3D subtomograms?

A

2 Euler angles
2 in-plane X/Y positions
1 Z-height (defocus)

31
Q

How can cryo-ET and STA be used in virology and cell biology?

A

To understand dynamic process = membrane fusion and cell entry
The entire virus or cell can be imaged instead of isolated components

32
Q

Is it possible to predict the structure based on stereochemistry?

A

For most proteins, this doesn’t YET work but possible for some very small proteins ~80 resides

Folding timescales are usually longer than simulation timescales
Disulphide bonds form during the real folding process = hard to mimic in simulation

33
Q

What are protein homologs?

A

They evolved from a common ancestor

Fold of the proteins tends to be conserved during evolution = so tend to have similar structure

34
Q

Structure or sequence are more conserved?

A

Structure = sequences change more than structure

35
Q

What does Alphafold do?

A

Predict structures from sequences

Protein Structure & Function (11 decks)

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