Imaging Techniques Flashcards
Why is it important to understand a protein’s 3D structure?
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
What resolution does single-particle cryo-EM look at?
Structural biology, near atomic resolutions so can see side chains and proteins
What resolution does cryo-electron tomography look at?
Proteins > macromolecular machines>bacteria
This is the gap between structural and cell biology
What are the uses of cryo-ET in infectious disease?***
Visualise pathogen structure and assembly
Can understand how the pathogen interacts with its target
What do cryo-ET subtomogram averaging and single particle cryo-EM look at?
Cryo-EM = whole cells, unique and heterogeneous
Single particle = purified macromolecules, isolated and homogeneous
What is the difference between resolutions for cryoET/EM?
Cryo-ET looks at the native and complex, resolution increases
Single-patricle = looks in vitro with high resolution
What is important in biological sample preparation?
Preserve the natural integrity
Thin layer of suspension
How is the natural integrity of the sample kept?
Hydrated at low temperatures and in a vacuum = no crystal ice forms and it is not dry
Inactivation for biosafety***
What is cryofixation?
Sample preparation method
How can they use such low temperatures but not have ice?***
Liquid ethane heat transfer much quicker
Water molecules don’t have time to crystallize
Explain manual vs automatic plunger for freezing?
Manual uses a steel rod to plunge the sample in quickly
Automatic plunger uses vitrobot
What are the different methods of cellular sample preparation?
Room-temp EM = high pressure freezing for thickness <200nm
Cryo-EM = plunge freezing for thickness <20nm
What happens when sample is thick?
Higher kV is needed
How does high pressure freezing (HPF) work?
Increased pressure means temp for freezing is reduced
At pressure 2045 bar = freezing temp -92
What is cryo-FIB milling?***
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
Why is the sample coated in metal for cryo-FIB?
To increase electron conductivity
What is a polyQ inclusion?
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.
What do we need to think about once the sample is ready to go?
Which microscope to use?
Which parameters to set up?
How to collect data?
How do you get 3D structure from TEM?
Electrons pass through the biological sample and the camera below records only 2D projections
How do you get 2D projection from different views?
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
What machine is used for high-resolution cryo-ET and subtomogram averaging?
Titan Krios = 300kV can penetrate thick samples
Direct Electron Detector(DED) w energy filter
Optimized stability to reduce drift
How are the samples on cryo-TEM screened?
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
What needs to be taken into account with tilt series?
Tilt range = completeness of information and sample thickness
Electron dose = signal-to-noise ratio and radiation damage
Angular increment = dose and signal distribution
What are the units for electron dose of sample?
e- / A^2
What is the tilt series scheme they established was most effective?
Dose-symmetric tilt series scheme
From -60 to +60 in 3 degree increments
What are the two ways of projection?
Forward projection = imaging
Back projection = reconstruction
What is subtomogram averaging?
Collection of subtomograms from one tomogram to give better resolution
How does subtomogram averaging work?
Particle picking
Lattice map
Subtomogram extraction and alignment
What are the steps for subtomogram extraction and alignment?
- Subtomograms = extracted from reconstructed tomograms
- Rotational and translational alignments are performed against a reference
- Aligned subtomograms are averaged to generate a new reference
- The new reference is used for iterative alignments
Procedures 1-4 are repeated until the reference stabilizes
How do they reconstruct a 3D structure from 2D projections or 3D subtomograms?
2 Euler angles
2 in-plane X/Y positions
1 Z-height (defocus)
How can cryo-ET and STA be used in virology and cell biology?
To understand dynamic process = membrane fusion and cell entry
The entire virus or cell can be imaged instead of isolated components
Is it possible to predict the structure based on stereochemistry?
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
What are protein homologs?
They evolved from a common ancestor
Fold of the proteins tends to be conserved during evolution = so tend to have similar structure
Structure or sequence are more conserved?
Structure = sequences change more than structure
What does Alphafold do?
Predict structures from sequences
