above, to be very similar. Unfortunately, particles usually have an
orientation bias, which arises from their adherence to one of two
(top and bottom) interfaces on a grid. These can either be an air–
water or support–water interface [19, 20]. Adherence to an interface results in an uneven distribution of particle projections. Consequently, the reconstruction of a 3D density is typically not evenly
resolved in all directions, and the directional resolution curves can
be very different [6]. Just as flexibility affects primarily local resolution, but not necessarily directional resolution, preferred orientation affects directional resolution, but not necessarily local
resolution. There are exceptions, for example, if preferred orientation also causes particle denaturation (in which case local resolution
would also be affected) [15], but such special cases are not
discussed here.
Here, we will describe protocols for calculating local and directional resolution for reconstructed density maps. Other than commenting on some general features of the curves, we do not describe
the calculation of the global resolution, which is standard in all
cryo-EM processing packages. Different samples will be presented
to show distinct aspects of resolution evaluation, including structural homo/heterogeneity and directional (an)isotropy. We will
provide general guidelines when evaluating the results from these
measures using experimental samples.
2 Materials
2.1 Software
1. Local resolution: SPARX [21] (see Note 1 about the use of
alternative softwares, e.g., Relion or CryoSparc). In order to
run local resolution estimation, the software described in the
current protocol can be downloaded from:
http://sparx-em.org/
2. Directional resolution: 3DFSC. In order to run directional
resolution estimation using the command line, the software
described in the current protocol can be downloaded from:
https://github.com/LyumkisLab/3DFSC
The software requires an installation of the Anaconda distribution (the scientific platform for Python), which can be
downloaded from:
https://www.anaconda.com
The web version of 3D FSC is available through a
web-server at:
https://3dfsc.salk.edu
3. Visualization: UCSF Chimera. This program allows visualizing
and analyzing the results from local and directional resolution
analysis. Chimera can be downloaded from:
https://www.cgl.ucsf.edu/chimera/
Local and Directional Resolution in Cryo-EM Maps
165
orientation bias, which arises from their adherence to one of two
(top and bottom) interfaces on a grid. These can either be an air–
water or support–water interface [19, 20]. Adherence to an interface results in an uneven distribution of particle projections. Consequently, the reconstruction of a 3D density is typically not evenly
resolved in all directions, and the directional resolution curves can
be very different [6]. Just as flexibility affects primarily local resolution, but not necessarily directional resolution, preferred orientation affects directional resolution, but not necessarily local
resolution. There are exceptions, for example, if preferred orientation also causes particle denaturation (in which case local resolution
would also be affected) [15], but such special cases are not
discussed here.
Here, we will describe protocols for calculating local and directional resolution for reconstructed density maps. Other than commenting on some general features of the curves, we do not describe
the calculation of the global resolution, which is standard in all
cryo-EM processing packages. Different samples will be presented
to show distinct aspects of resolution evaluation, including structural homo/heterogeneity and directional (an)isotropy. We will
provide general guidelines when evaluating the results from these
measures using experimental samples.
2 Materials
2.1 Software
1. Local resolution: SPARX [21] (see Note 1 about the use of
alternative softwares, e.g., Relion or CryoSparc). In order to
run local resolution estimation, the software described in the
current protocol can be downloaded from:
http://sparx-em.org/
2. Directional resolution: 3DFSC. In order to run directional
resolution estimation using the command line, the software
described in the current protocol can be downloaded from:
https://github.com/LyumkisLab/3DFSC
The software requires an installation of the Anaconda distribution (the scientific platform for Python), which can be
downloaded from:
https://www.anaconda.com
The web version of 3D FSC is available through a
web-server at:
https://3dfsc.salk.edu
3. Visualization: UCSF Chimera. This program allows visualizing
and analyzing the results from local and directional resolution
analysis. Chimera can be downloaded from:
https://www.cgl.ucsf.edu/chimera/
Local and Directional Resolution in Cryo-EM Maps
165