5 - Cryo EM - Egbert Flashcards
what are the 3 pillars of structural biology? name what they are used to study
X-ray; protein crystal
NMR; protein solutions
Cryo EM; organisms, cells, protein suspensions
state what the resolutions are for each of the 3 pillars of strucutral biology
X-ray; atomic res. Measure proteins -> complete viruses
NMR; atomic res. small proteins and protein domains
Cryo EM; thin section - 5nm, -ve stain - 2nm, ice embedded - atomic res
what are the downsides of X-ray crystallography and NMR?
X-ray; need symetrical structures eg cannot use X-ray to determine bacteriophage structure because not symmetrical
NMR; needs a high protein conc which needs to be in liquid (some proteins this isnt possible). a lot of proteins are too large therefore inaccessible for NMR
what does the image in q4 328 - 5 word show?
negative stain image obtained using cryoEM of a proteasome
draw a diagram and explain the physical basis of electron imaging ie how different beams can be scattered depending on what part of the e- they hit. state what parts of the e- each beam hits
INELASTIC SCATTERING; causes a loss of energy and change in wavelength. used for spectroscopic analysis. this interacts with the electron cloud
ELASTIC SCATTERING; does not reuslt in change of energy or wavelength. used for structural analysis and therefore used in imaging. this beam interacts with nucleus
what kind of damage does the electron beam cause to the sample? what are the effects of this?
causes radiation damage when the e- transfer their energy onto the specimen -> molecular ionisation and radiolysis (bond dissociation) resulting in molecular ions and free radicals invovled in 2ndry reactions. therefore limiting the overall resolution
why has the no. structures solved by NMR declined over the past decade?
only used to resolve small structures , majority of these have already been solved
why is EM being used over Xray crystallography to determine membrne protein strucutre?
becauuse membrane proteins are in lipid environemnt , cannot crystallise them. cryoEM does not require crystallisation of the protein
what is responsible for the sudden success of EM in strucutral biology?
- introduction of direct electron detectors; allows us to compensate for the movement of molecules during beam exposure. improving quality of data collected
- improvements in classification and alignment procedures combined with increasing power of computers allows for large data sets to be processed
- improvements in stability of specimen holders and coherence of e- beam
how do direct e- detectors work and what do they do? draw a diagram of this process
- allow us to see images at a much higher resolution
- record the imaging, look at the individual frams for movement, realignment and creating an average, creation of a high res image
328 - 5 word
give an example of a structure that direct electron detetors have allowed us to visualise clearly
rotavirus particles (328 - 5 word)
What are the advantages and disadvantages of electon microscopy?
ADVANTAGES;
- does not require crystalisation
- samples do not need to be highly pure - as long as we have correct alignment and classification procedures
- can solve structure of large complexes (eg viral capsids, ribosomes) and membrane proteins (require detergents to keep structural integrity)
- does not have a phase problem (phase problem = eg in X-ray we need a reference point with a heavy atom)
- can resolve complex conformational changes
DISADVANTAGES;
- e- beam only works in a vacuum
- radiation damage to specimen from e- beam
- proteins smaller than 100kDa hard to resolve
- small cells eg bacteria are often too large to be viewed in toto
- large scale dynamics are difficult to capture
- validation may be difficult. how do we know that results are correct?
draw a diagram to highlight the plunge freezing process.
state why it is difficult to identify smaller structures once they have been frozen
- problem = low contrast. even computer cannot determine between the ice crystals and the sample
draw a diagram and explain how we would achieve a 3d image of a certain protein following alignment procedures etc
- multiple frames obtained and aligned to create a summed movie frame
- computer picks out the raw particles
- raw particles then evaluated and averaged into 2D structures
- initial model generated using these 2d structures
- high res 3D map then generated by combining new particles aligned to the refernce
- we now have a high resolution 3d map atomic model
what is tomography and what type of image does it produce? give an example of a particle that tomography can image. what is a downside to tomography?
multiple angles and shifts of the same specimen used to build up a 3d image eg HIV particle
- produces 3d image
- -ve; cannot easily achieve atomic resolution
