Samenvatting EM HC.8
Single particle analysis
If we have 2D projections from all directions we can reconstruct the 3D object.
The data collection for this
approach consists in taking 2D
projections of a sample containing
many identical but differently
oriented copies of the same object
Low signal to noise ratio (SNR) in raw micrographs (nowadays very good algoritms)
Need to merge thousands of 2D projections corresponding to a similar direction to
generate an averaged image of that direction (and reduce the noice)
Need to have all directions to reconstruct a 3D structure
Good sample preperation is crucial. You want:
Pure samples (no contaminants)
Limited compositional conformation variability
Stable enough to handle grid preperation
Well preserved in thin and vitreous ice
Plunge freezing can be very harsh for (fragile
proteins). First of all many proteins are unstable
outside a cell. When the protein is exposed to a
hydrophobic air-water interface (which is
hydrophobic), it is competing for the folding of the
proteins and that can result in the unfolding of the
proteins.
, There are different things you can do to avoid the air-water interface (prevent denaturation):
Thin carbon support across the holes (< 2nm)
Graphene (single atomic layer)
Surfactants or detergents to obscure interface
They add a little bit of background, but your proteins are safe
CryoEM raw micrographs have very low signal to noise ratio. More mass (bigger particles)
means more signal and a higher signal to noise ratio. So for SPA you need a lot of images! To
get so many imaged they make use of a automated data collection. Patterning grids with
regular holes facilitates data acquisition and sample consistency.
Challenge SPA beam induced motion. The beam exposure causes
the sample to move. Why this happens is not clearly known yet. It could
be that there is an increased pressure due to beam damage or there is
charging. The frustrating thing is that motion is the worst at the
beginning of the exposure, when the radiation damage is the least.
Movies are critical for a good alignment. You can correct for rotations,
translations and defocusing.
B-factor: weighting used to determine the contribution of the
frame to the final reconstruction. In the beginning there is less
contribution due to motion and on the end due to radiation
damage.
Single particle analysis
If we have 2D projections from all directions we can reconstruct the 3D object.
The data collection for this
approach consists in taking 2D
projections of a sample containing
many identical but differently
oriented copies of the same object
Low signal to noise ratio (SNR) in raw micrographs (nowadays very good algoritms)
Need to merge thousands of 2D projections corresponding to a similar direction to
generate an averaged image of that direction (and reduce the noice)
Need to have all directions to reconstruct a 3D structure
Good sample preperation is crucial. You want:
Pure samples (no contaminants)
Limited compositional conformation variability
Stable enough to handle grid preperation
Well preserved in thin and vitreous ice
Plunge freezing can be very harsh for (fragile
proteins). First of all many proteins are unstable
outside a cell. When the protein is exposed to a
hydrophobic air-water interface (which is
hydrophobic), it is competing for the folding of the
proteins and that can result in the unfolding of the
proteins.
, There are different things you can do to avoid the air-water interface (prevent denaturation):
Thin carbon support across the holes (< 2nm)
Graphene (single atomic layer)
Surfactants or detergents to obscure interface
They add a little bit of background, but your proteins are safe
CryoEM raw micrographs have very low signal to noise ratio. More mass (bigger particles)
means more signal and a higher signal to noise ratio. So for SPA you need a lot of images! To
get so many imaged they make use of a automated data collection. Patterning grids with
regular holes facilitates data acquisition and sample consistency.
Challenge SPA beam induced motion. The beam exposure causes
the sample to move. Why this happens is not clearly known yet. It could
be that there is an increased pressure due to beam damage or there is
charging. The frustrating thing is that motion is the worst at the
beginning of the exposure, when the radiation damage is the least.
Movies are critical for a good alignment. You can correct for rotations,
translations and defocusing.
B-factor: weighting used to determine the contribution of the
frame to the final reconstruction. In the beginning there is less
contribution due to motion and on the end due to radiation
damage.
