CryoEM and TEM Flashcards
What resolution advantages does TEM have over optical microscopes?
TEM has resolution of 0.2nm vs optical microscope resolution of 200nm
So TEM can view structures at atomic resolution, revealing structure of protein folding as opposed to optical microscopes at micron resolution which show whole cells/tissues
What is the resolution principle?
Resolution = closest spacing of two points that can be defined by the microscope to be seperated entities
Resolution is limited by wavelength; specimen must be imaged with a wavelength in range (or smaller than) the target image to avoid blurring
What is the advanhtage of using electrons (TEM) rather than waves of light (optical microscope)?
wavelengths of accelerated electrons much shorter than light - this increases resolution since resolution is limted by wavelength.
electrons do behave like wavelengths of light (scattering) but with higher penetration –> formation of airy disk
Give an example of how Crystallography using TEMs has developed to reveal atomic structures of 3.3 Angstrom with CryoEM?
Clue - toxins
Firstly Specialised Electron Detectors have faster readout, higher detective quantum efficiency and no image distortion. This is opposed to the original Scintilator cameras used in TEM which were not designed for imagine electrons; caused splitting so noisy images.
So improving the capture time/speed was a big contributor.
Secondly high resolution data was capturable using nanodisks, inserted into side entry holders. This made the sample more stable. Membrane proteins are particularly hard to crystallise; nanodiscs overcame this and allowed for the visualisation of interacting phospholipids. For example, Stefan Raunser at the Max-Planck Institute in Germany demonstrated the mechanism by which bacterial Tc toxin enters its host cell (these toxins include plague and scarlet-like fever toxins - so highly relevant to medical field). Previous attempts with detegent-solubilized protein could not resolve the transmembrane region of the protein, but using nanodisc-stabilized TcdA1 protein researchers reached an overall Angstrom of 3.5.
Outline Negative Staining method and what it might be used for.
- apply small drop of dry sample on carbon support film, this is placed on an EM grid
- apply heavy metal stain (uranyl acetate) –> this forms a capsid around the particle which will cause scattering of the beam, increasing the contrast of the image.
- blot and air dry
- image at room temp –> you get a 2D projection of all the protein in your 3D sample
Good for visualising size and shapes of molecules, such as the Adenovirus. Also immunogold labelling is easy in negative staining, can use this to visualise extracellular vecles in glioblastoma cancer - finding treatments.
How do you decrease the signal:noise ratio of Negative Staining TEM images?
2D SPA (single particle reconstruction) –> averaging multiple images of a similar view to make a 3D construction. Angles are correlated by cross-correlation (computer programs). This helps you construct protein assembly - subunit info eg numbers and arrangements
The images come from the uniform spread of particles (stained with negative stain) uniformly across an EM frid in the form of thin film.
Why might you do negative staining before cryoEM?
to test the stability of the sample and reveal low resolution 3D structure of trhe macromolecule –> can assess how suitable the sample is for high res 3D CEM
Outline the CryoEM methods.
first do a diagnostic -ve staining to see stability of proteins.
freeze the sample in vitrous ice to visualise in its native state (in negative staining, the removal of water affects the structure of proteins) –> sample prep
sample spread into thin film over EM grid, blotted to remove excess buffer (for effective freezihng), then embedded in vitrous ice by plunge freezing in liquid ethane (-170 deg)
The grid preparation is important. electron microscope grids 3mm coated in carbon film and perforated with holes - the proteins of interest become embedded in these holes once vitrified. Ice crystal formation prevented by maintaing the sample at -160 deg.
CryoEM data collection of homologous images
Then high res CryoEM using 3D SPA to construct conformational states and overall resolution
compare negative staining with cryoEM
cryoEM:
sample is hydrated and stabilized in vitrous ice (plunge freezing in liquid ethane) –> lower signal:noise ratio so can visualise higher resolution
sample kept at -160degs
negative staining:
imaging at room temp
lower resolution but higher sig:noise (enhances contrast) - due to heavy metal cast
sample is dehydrated, this can cause artefacts
How do you get a 3D Cryo-EM image?
volume reconstruction using back projection (3D SPA) you combine similar angles of loads of 2D images to construct the 3D image. These need to be homogenous (alligned)
