Lecture 5: Methods Flashcards
(39 cards)
What is a big advantage of Cryoelectromicroscopy depicted here:
- There multitude subtechniques used within it -> allows for a huge range of small structures
What needs to be done before we even start using cryoEM?
Sample preparation and Grid freezing
- the sample is placed onto a grid held with tweezers
- this already differs based on the particles (e.g. how dense they are, what kind of buffers we apply)
-> blotting: removing excess liquid
-> put our grid into liquid ethane bath in order to freeze it as quickly as possible
Frozen grid gets loaded into specific machines -> look at how the grid squeres looks like
Sample found in the holes in the grid squeres
- Still depends on quality of the sample - if good, computer can pick it up and perform analysis by itself, if not some manual selection has to be done
What is the next step?
Image processing
- particles are frozen in all kinds of positions -> pick individual particles (computer or manually) -> 2D classified - clustering similarly looking particles -> getting images in all perspectives and create 3D reconstruction with those
- Resolution achieved by imaging over a lot of particles
-> we can see similarities and differences between particles
NOTE: advancements
- more cameras
What kind of advancements were made thus far?
- Improving electron detection cameras
- Imaging processing got cheaper and more accessible
=> solving even smaller molecules, even protons
NOTE: stay critical - how are claims of experiments backed up
How does light microscope works (very simplistically)? Why do we not use it for proteins?
Light hits the object of interest -> gets reflected from it and arrives at the Objective lens -> magnification of the original -> reflected into our eyes
- Works because the electromagnetic radiation wavelength (visible light) is in the same scale as our object of interest (microscopic scale)
- well suited for studying micrometer to milimiter sized objects
- BUT if we want to visualize proteins we need different radiation - X-ray radiation
Why is it still not possible to get an image of an atom or protein from a single molecule? How can we resolve this?
1) X-ray cannot be focused by lenses - principles used in light microscope don’t work here
2) Single molecule creates a weak scatter of X-rays (only small portion gets refracted - most pass through)
-> however we can crystalize the protein to resolve this
So what components do we need for this method?
What is the workflow process step by step?
What is meant by protein crystals here? How is it different from inorganic crystals?
= regular array of individual protein molecules stabilized by crystal contacts
- high water content
- few crystal contacts (both unlike inorganic crystals)
- retain their function e.g. enzyme activity, ligand binding
What are asymmetrical unit or unit cell? What kind of operations are the most common?
- Asymmetric unit = crystallographic unit cell which can be used to generate the complete unit cell by the symmetry of the space group
- Unit cell = simplest repeating unit in the crystal
- Operations: rotation, translation, their combination
- E.g. rotation of the assymetric unit results in the red structure -> together with original creates a unit cell -> translated into generates entire crystal
Look at examples of crystals.
Depending on crystalization conditions the same proteins may crystalize differently
How do we recognize good X bad crystals?
Good crystals = difract well
- well formed, sharp edges
- no umpurities
Bad crystals = bad refractions
- some impurity compromises defraction
- instead of growing in one direction they form mosaic misorientation
-> additional data processing steps would need to be applied (still usually avoided)
What is the first step of making such crystals?
1) Design constructs wisely
- Add affinity tags (e.g. His6)
- they improve and speed up purification
- put it at the beginning or end of the protein
- removed by protease (if we think it affects the function of the protein)
- We can add mutations
- if we want to study inactive protein
- or stabilize it
- or if sample is hetergenous
- We need correct light conditions
What is the second step of making such crystals?
2) Protein expression and purification
- We tend to use some cloning strategies to get more of the protein = protein expression
-> Purification - using size, charge (exposure to salt gradient) or affinity to a specific molecule to “trap” the desired protein and get rid of the rest
- varies the movement of proteins through a gel
- depending on the protein we need to choose the method that would exclude the most efficiently e.g. if protein is hollow we might need charge instead of size
-> Dialysis
- change of the buffer of concentration
-> Concentration
- usually quite low -> we need to concentrate/accumulate to get enough for crystalization
What is the general principle of crystalization?
Put protein in aquatious bath with precipitant -> concentration of the protein and the precipitant is just below the precipitation process -> slowly increase concentration of both (slow controlled evaporation) -> we reach precipitation zone -> controlled aggregation of the protein -> forming crystaks
In actuality it tends to be more tricky:
Starting condition -> precipitation reached -> aggregation -> protein concentration drops (while precipitant remains) -> drop into nucleation zone = ordered clustered proteins -> nucleation forming while evaporation continues -> protein concentration decreases -> drop into metastable zone = crystals keep growing, all the remaining proteins will end up as crystals = less but bigger crystals (as opposed to many but small) -> that’s more ideal as they refract radiation better
How does the vapur diffusion/evaporation work?
Hanging drops - drops of proteins are hanging above the wel
Sitting drops - put at a specialized position
-> we put high concentration of precipitant into reservoir wel -> take tiny drop of solution and mix it with drop of the protein -> decrease precipitant and also protein concentration twice -> seal the wel -> concentration of precipitant in the drop of protein will start equilibrate concentration of the percipitant in the wel until their balanced because of water slowly evaporating -> increasing concentration of proteins and percipitant in the drop
Look at proteins in different phases of crystalization:
What is special (and relevant) about X-ray?
How is it generated?
= electromagnetic radiation which, thanks to its short wavelengthcan interact with electrons of the matter (so also ptoteins)
- the wavelengths are compatible with separation between atoms in compounds -> can be used to resolve questions of structure (through electron density)
- Generated by accelaration or deaccelarating electrons
- about 90% of applied X-ray will be transmitted, only 8% difracted
What causes diffraction of X-ray in crystalized proteins?
X ray shines on the crystallized protein -> beam interacts with electrons -> scatter
-> either destructive interference = scattered beams cancel each other out (lower amlitude)
-> constructive interference = beams reinforce themselves (higher amplitude) -> project specific pattern = Bragg spots that can be detected -> careful computation builds the structure
-> as we rotate the crystal we can get more info on electron density (-> and structure of atoms) - constructed by FT
What is Bragg equation?
=> equation that quantifies constructive interference
We have two lattice planes -> two atoms A and B with distance D
-> they will be in phase (constructive interference) if the extra distance travelled by an X-ray scatter byD is a whole number of wavelength
-> at regular lattices (crystals) we get positive interference when wavelength and lattice distance are in the same order of magnitude and exactly obey Bragg’s law
What does the technical setup contains?
What may X-ray source contain?
- K-alpha radiation = source
- Catode and anode in vaccum -> catode contains filament that gets heated up ->loosing electrons (being accelerated) -> hit rotating anode -> electrons go from inner shell to outer shell -> in order to go back to inner shell they need to emit photon of electromagnetic energy (K-alpha transition)
