Fig. 2c). We have also observed that severe preferential orientation can also apparently skew the local resolution values, as is
the case with the reconstruction of HA from untilted images
described in the current protocol. However, we note that local
and directional resolution measurements provide fundamentally distinct assessments of the map, and a detailed analysis as
to the origin of this phenomenon is beyond the scope of the
current protocol. It is always worthwhile to carefully inspect
the map and the structural features within every local resolution patch.
8. In general, the values for local resolution should vary slightly
around the global resolution of the volume, as estimated by the
global FSC (the extent can vary). If this is not the case, then
something in the protocol may be wrong, and the user should
inspect the inputs. A common mistake is to not set the proper
values for the voxel size or the origin.
9. There have been several implementations of the conical resolution estimates applied to tomographic reconstructions [16, 17]
and several implementations applied to single-particle reconstructions [6, 35]. These measures are all similar conceptually,
with minor variations in how the data is weighted and/or how
the cone is defined.
10. For more options, the user is directed to the help documentation of the standalone 3DFSC program.
11. Computing the 3D FSC requires two half-maps to calculate
the half-map FSC. In order to calculate map-to-model FSC, a
map has to be generated from a model derived from the
density. In the absence of such a model, the map-to-model
FSC cannot be computed. In order to generate a map from a
model for the methods described here, the e2pdb2mrc command from the EMAN [36] software package was used, and
the map was computed at a resolution corresponding to
Nyquist frequency. For example, the command used to obtain
a synthetic map from the AAV2 model (PDB ID 6E9D) was:t
>> e2pdb2mrc.py 6E9D.pdb 6E9D.mrc --apix 0.79 --res 1.58 -box 512
There are other methods to calculate a molecular model,
for example, in Chimera [14], or in other software packages.
Once the map is generated, it must be aligned to match the
dimension and origin of the experimental map to be used for
analysis. This can be achieved in Chimera by resampling the
map generated from the model onto the coordinates of the
experimental map.
Local and Directional Resolution in Cryo-EM Maps
181
the case with the reconstruction of HA from untilted images
described in the current protocol. However, we note that local
and directional resolution measurements provide fundamentally distinct assessments of the map, and a detailed analysis as
to the origin of this phenomenon is beyond the scope of the
current protocol. It is always worthwhile to carefully inspect
the map and the structural features within every local resolution patch.
8. In general, the values for local resolution should vary slightly
around the global resolution of the volume, as estimated by the
global FSC (the extent can vary). If this is not the case, then
something in the protocol may be wrong, and the user should
inspect the inputs. A common mistake is to not set the proper
values for the voxel size or the origin.
9. There have been several implementations of the conical resolution estimates applied to tomographic reconstructions [16, 17]
and several implementations applied to single-particle reconstructions [6, 35]. These measures are all similar conceptually,
with minor variations in how the data is weighted and/or how
the cone is defined.
10. For more options, the user is directed to the help documentation of the standalone 3DFSC program.
11. Computing the 3D FSC requires two half-maps to calculate
the half-map FSC. In order to calculate map-to-model FSC, a
map has to be generated from a model derived from the
density. In the absence of such a model, the map-to-model
FSC cannot be computed. In order to generate a map from a
model for the methods described here, the e2pdb2mrc command from the EMAN [36] software package was used, and
the map was computed at a resolution corresponding to
Nyquist frequency. For example, the command used to obtain
a synthetic map from the AAV2 model (PDB ID 6E9D) was:t
>> e2pdb2mrc.py 6E9D.pdb 6E9D.mrc --apix 0.79 --res 1.58 -box 512
There are other methods to calculate a molecular model,
for example, in Chimera [14], or in other software packages.
Once the map is generated, it must be aligned to match the
dimension and origin of the experimental map to be used for
analysis. This can be achieved in Chimera by resampling the
map generated from the model onto the coordinates of the
experimental map.
Local and Directional Resolution in Cryo-EM Maps
181