and recently by Chen and co-workers [98]. There are some general guidelines [80,
99] suggested, and they are actively evolving. As described in the resolution paragraph of this section, the reporting of resolution with one number cannot be used as
validation; however, it is an important parameter to be reported during EMDB
deposition. Further, FSC may fail when the particles are significantly misaligned. So
one has to estimate the resolution properly [76] and use the reported single number
resolution with caution. It is suggested that gold-standard FSC provides a realistic
estimate of the true signal [100], and this will lead ultimately to a better map. In
recent days, reporting local resolution has also formed a common practice in publication and thesis [81, 82]. Also, the local resolution will be helpful in avoiding
over-interpretation of poor regions in the cryo-EM map. If the 3D map is of sufficient
resolution (better than 4 Å), it can resolve the secondary structural features. A good
validation would be especially if you can see a right-handed alpha helix or even the
side chain residues, especially the bulky residues like tryptophan, phenylalanine, or
tyrosine in the high-resolution cryo-EM map. Even the comparison of the new EM
structure with the available EM structure will be one way of validating the newly
reconstructed 3D EM map [101]. Further, programs TEMPy [102] and refmac [88]
can be used to assess the validity of the fitted coordinates to the EM map. In Coot
[86] program, one can use the Ramachandran plot (Validate !Ramachandran plot)
and geometrical quality (Validate !Geometry analysis) to validate the quality of the
refined model. One more way to validate is to compare the 3D reconstruction results
from different techniques, e.g., projection matching and the angular reconstitution.
For low-resolution maps (worse than 4 or 10 Å), measure of confidence can be
provided by a priori random conical tilt experiments [103].
4 Heterogeneity
Though cryo-EM can handle heterogeneous particles, we need homogenous particles, which are equally dispersed in the vitrified ice in order to achieve atomic
resolution. Ideally speaking, all data sets are heterogeneous! The question is how
much one is willing to tolerate [104]. Further, during cryo-EM specimen preparation, non-physiological structural heterogeneity is often introduced [105]. While
structural heterogeneity is a problem to obtain high resolution, it also provides a
unique opportunity to study the conformational flexibility/dynamics of the
macromolecular assemblies. Ideally, homogenous samples have to be biochemically standardized and prepared before the vitrification process. However, this is not
possible with all protein samples due to the inherent protein flexibility which is
necessary for its function, for example, rotation of 30S subunit of ribosome [106] or
rotational states in case of eukaryotic V-ATPase [107]. In such cases, the heterogeneous sample data images can be classified computationally to classes containing
homogenous particles (an example of such classification can be seen in Fig. 4).
Three main techniques are currently in use to identify and sort the macromolecular structural conformational variability or heterogeneity [41]. The first
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
391
99] suggested, and they are actively evolving. As described in the resolution paragraph of this section, the reporting of resolution with one number cannot be used as
validation; however, it is an important parameter to be reported during EMDB
deposition. Further, FSC may fail when the particles are significantly misaligned. So
one has to estimate the resolution properly [76] and use the reported single number
resolution with caution. It is suggested that gold-standard FSC provides a realistic
estimate of the true signal [100], and this will lead ultimately to a better map. In
recent days, reporting local resolution has also formed a common practice in publication and thesis [81, 82]. Also, the local resolution will be helpful in avoiding
over-interpretation of poor regions in the cryo-EM map. If the 3D map is of sufficient
resolution (better than 4 Å), it can resolve the secondary structural features. A good
validation would be especially if you can see a right-handed alpha helix or even the
side chain residues, especially the bulky residues like tryptophan, phenylalanine, or
tyrosine in the high-resolution cryo-EM map. Even the comparison of the new EM
structure with the available EM structure will be one way of validating the newly
reconstructed 3D EM map [101]. Further, programs TEMPy [102] and refmac [88]
can be used to assess the validity of the fitted coordinates to the EM map. In Coot
[86] program, one can use the Ramachandran plot (Validate !Ramachandran plot)
and geometrical quality (Validate !Geometry analysis) to validate the quality of the
refined model. One more way to validate is to compare the 3D reconstruction results
from different techniques, e.g., projection matching and the angular reconstitution.
For low-resolution maps (worse than 4 or 10 Å), measure of confidence can be
provided by a priori random conical tilt experiments [103].
4 Heterogeneity
Though cryo-EM can handle heterogeneous particles, we need homogenous particles, which are equally dispersed in the vitrified ice in order to achieve atomic
resolution. Ideally speaking, all data sets are heterogeneous! The question is how
much one is willing to tolerate [104]. Further, during cryo-EM specimen preparation, non-physiological structural heterogeneity is often introduced [105]. While
structural heterogeneity is a problem to obtain high resolution, it also provides a
unique opportunity to study the conformational flexibility/dynamics of the
macromolecular assemblies. Ideally, homogenous samples have to be biochemically standardized and prepared before the vitrification process. However, this is not
possible with all protein samples due to the inherent protein flexibility which is
necessary for its function, for example, rotation of 30S subunit of ribosome [106] or
rotational states in case of eukaryotic V-ATPase [107]. In such cases, the heterogeneous sample data images can be classified computationally to classes containing
homogenous particles (an example of such classification can be seen in Fig. 4).
Three main techniques are currently in use to identify and sort the macromolecular structural conformational variability or heterogeneity [41]. The first
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
391