Berger_cryoEM Flashcards
why can’t you use light to image molecules?
limit of resolution obtainable by light is around 300 nm
why do different light resolution limits exist in the literature?
because the minimum distance needed to distinguish two point sources from one another is a matter of definition
why use electrons to image?
they’re both particles and waves, and they’re stupid small with very short wavelengths capable of given you much higher resolution
how do most electron detectors now work?
CMOS sensor, direct electron transfer/readout, works from the mvmt of electrons through a photodiode, amplified and conducted to an output
what are some things that mess with resolution (8)?
electron energy, optical aberrations, focus setting, low sample contrast, low signal to noise, radiation damage, detector quality and sample heterogeneity
what is negative stain EM?
most common, purify protein and apply to grids, apply U(OAc)2 and blot
what are 4 benefits of negative staining?
good signal to noise, fast/cheap, smaller particles can see, and lower concentrations needed
what are 4 cons of negative staining?
can induced preferred orientation, may cause artifacts/molecule collapse, has limited resolution and can’t see nucleic acids really
what are 4 benefits of cryoEM?
protein is in native state, higher resolution possible, more conformations possible, and can see nucleic acids
what are 4 cons of cryoEM?
low signal to noise, technically challenging, sample needed to be bigger and more concentrated
what is a way to stabilize weak complexes or trap intermediate configurations?
cross linkers
why are gold grids better?
they reduce beam induced movement by conducting electrons away from the sample rapidly
how many particles do you try to image for a dataset?
100K - 1M
what is a strategy for 3D refinement?
projection matching, you take class averages of different conformations, use them to build a 3D model, then do re-projections, compare to class averages, and iterate until they don’t change
why would you choose to use domain or subunit masking?
this way you can identify distinct conformations and improve particle alignments with better resolution
