Lecture 7: Electron Microscopy and Single Molecule Methods Flashcards
What is transmission electron microscopy?
TEM is a method which is used to study macromolecules and complexes by using beams of electrons.
• In TEM, a beam of electrons passes through the sample and we gain information based on their interactions.
• Biological samples normally have very low contrast. There is a low signal to noise ratio. We need to stain in order to correct for this.
• The stain covers the sample (positive staining with uranyl acetate) or the background (negative staining). Staining causes distortion from fixation dehydration and staining.
• For negative staining, the electrons go through the protein more easily than the metal. The protein appears as a white silhouette.
• Rotary shadowing is when a metal is sprayed onto molecules which are adsorbed on a surface. Rotation gives more even exposure. The signal is good, but resolution is low.
• Traditional metal staining techniques give a resolution limit of 2 nm or worse.
What is cryo electron microscopy?
Cryo-EM can massively improve resolution up to about 0.2 nm.
• Radiation damage is minimised from the cooling of the sample.
• Use a grid to lower the water. Thin layer of carbon with regular holes. Hundreds of proteins in each hole.
• Lower the temperature to form vitreous ice.
• Fire electrons through the sample until it hits the detector.
What is electron tomography?
Tomography is used to find images of large cellular structures.
• We can perform single particle cryo EM with electron tomography.
• You get lots of images of different orientations.
• Sort images into groups and average them together.
• Add the 2D images together to get a 3D structure.
• Common line approach. Look for common lines of electron density. Computerised.
• Resolution is 3-10 nm.
• Some angles are inaccessible.
• ET was used to find the structures of 80S ribosomes.
• Another experiment found the structure of β-galactosidase at a 2.2 angstrom resolution (comparable to XRC).
How does electron crystallography work?
Electron crystallography can be used to find the arrangement of atoms in solids with a TEM.
• Electron crystallography gives an atomic resolution (around 0.2 nm).
• The sample has to be ordered. For example, a helical array or 2D crystals (proteins in a lipid bilayer).
• Many molecules contribute, so the signal/noise ratio is higher than single particle EM.
• Sample tilting (similar to rotating a crystal) increases data completeness.
• We encounter the phase problem, which can be solved by using tomography images.
• It was also used to find a 0.2 nm resolution of an aquaporin.
What is scanning electron microscopy?
In contrast to TEM, SEM can be used to create a 3D structure of a surface.
• An electron beam hits the sample.
• The electrons are scattered by the sample and they it a detector of secondary electrons.
• Resolution is lower than TEM.
• The sample is sputtered with gold.
• The images have good depth information.
• SEM can also be used to analyse the atomic composition of the sample.
What are the pros and cons of electron microscopy?
Pros
• No crystallisation.
• Captures the proteins as they are.
• You can look at different conformations. Group
• Can deal with some forms of flexibility. You couldn’t look at two parts linked by a flexible loop.
• Electron tomography will be a big advancement. Take lots of images of one particle at different angles.
• Can be used for viruses, actin dynamics, protein complexes.
Cons
• Low signal to noise ratio. Averaging images increases signal to noise ratio.
• Difficult to get the point where you have nice images.
• Doesn’t work for anything novel under 150 kDa. You can find smaller structures, but you have to cheat with existing structures.
What are single molecule experiments? Why are they useful?
Single molecule experiments allow us to look at one molecule at a time. This is contrast to most techniques, which give an averaged result of all the molecules.
• They require a high signal/noise ratio.
What is atomic force microscopy?
Atomic force microscopy can be used to look at the surface topologies of proteins.
• A sharp tip is scanned over the surface of a specimen to give an image.
• Resolution is fractions of a nanometre.
• It provides a 3D scan.
• It does take a long time to scan an object (several minutes) compared to SEM.
How can we trap or move particles?
Optical tweezers or magnetic traps can be used to trap or move a particle.
• A change in the direction of light due to scattering results in a change in momentum. This generates a small but useful force.
• In a light gradient, particles with a suitable refractive index bend light.
• The particle then experiences a net force form the EM radiation (around 10 pN) which can then be used to trap and/or move the particle.
• This can be used – for example – to immobilise an actin filament. The position of the beads can be measured with the accuracy of a few nm. We can then measure the displacement induced by actin.
• Magnetic traps can also manipulate single molecules. A magnetic bead was used to drive the rotation of ATP synthesis. It was linked to the gamma subunit using biotin, streptavidin and BSA.
