2.2 Cryo-EM Maps
1. Fullmap. This is the final reconstruction derived from the cryoEM experiment.
2. Half-map #1. First reconstruction from one half of the data.
3. Half-map #2. Second reconstruction from second half of
the data.
4. Map derived from PDB model. Density map computed from a
fitted molecular model (optional, and only for map-to-model
FSC calculations).
5. Mask. Used to remove solvent regions dominated by noise
(optional).
3 Methods
Subheading 3.1 will describe the protocol for local resolution
analysis. Subheading 3.2 will describe the protocol for directional
resolution analysis. Subheading 3.3 will provide the results of these
steps and analyses.
3.1 Estimating
and Evaluating
the Local Resolution
of a
Reconstructed Map
One of the first implementations of a measurement for evaluating
local resolution in cryo-EM based on conventional FSC analysis
was the blocres program [4] within the Bsoft processing package
[22]. There are now several other alternatives that have been
described in the literature (see Note 1). To estimate the local
resolution, we use the program sxlocres.py, which is also based on
FSC analysis. Because we have used this program for most of our
published structures, we adhere to its description, although many
programs offer similar implementations (see Note 1). This algorithm is implemented within the SPARX [21] package and
distributed with EMAN2 [23]. In adhering to conventional
FSC-based methods, we focus on describing this method here.
The resolution is computed in 3D patches windowed across the
map, and the nominal resolution value within the windowed region
is assigned to the centroid of the patch. The output is an identically
sized local resolution map, wherein a value is assigned to each voxel
within the density and each voxel corresponds to the local resolution value of the specific region of the input map. The evaluation of
the output from local resolution analysis is generally similar across
packages.
1. Navigate to the directory where both half-maps are located.
Optionally, you may wish to generate a mask to be used for the
analysis (see Notes 2 and 3).
2. Run a command, similar to the following, within the folder
that contains both half-maps.
>> /path/to/sxlocres.py /path/to/halfmap1.mrc /path/to/halfmap2.mrc local_resolution_volume.mrc --cutoff=0.143 --fsc=resolution.txt
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1. Fullmap. This is the final reconstruction derived from the cryoEM experiment.
2. Half-map #1. First reconstruction from one half of the data.
3. Half-map #2. Second reconstruction from second half of
the data.
4. Map derived from PDB model. Density map computed from a
fitted molecular model (optional, and only for map-to-model
FSC calculations).
5. Mask. Used to remove solvent regions dominated by noise
(optional).
3 Methods
Subheading 3.1 will describe the protocol for local resolution
analysis. Subheading 3.2 will describe the protocol for directional
resolution analysis. Subheading 3.3 will provide the results of these
steps and analyses.
3.1 Estimating
and Evaluating
the Local Resolution
of a
Reconstructed Map
One of the first implementations of a measurement for evaluating
local resolution in cryo-EM based on conventional FSC analysis
was the blocres program [4] within the Bsoft processing package
[22]. There are now several other alternatives that have been
described in the literature (see Note 1). To estimate the local
resolution, we use the program sxlocres.py, which is also based on
FSC analysis. Because we have used this program for most of our
published structures, we adhere to its description, although many
programs offer similar implementations (see Note 1). This algorithm is implemented within the SPARX [21] package and
distributed with EMAN2 [23]. In adhering to conventional
FSC-based methods, we focus on describing this method here.
The resolution is computed in 3D patches windowed across the
map, and the nominal resolution value within the windowed region
is assigned to the centroid of the patch. The output is an identically
sized local resolution map, wherein a value is assigned to each voxel
within the density and each voxel corresponds to the local resolution value of the specific region of the input map. The evaluation of
the output from local resolution analysis is generally similar across
packages.
1. Navigate to the directory where both half-maps are located.
Optionally, you may wish to generate a mask to be used for the
analysis (see Notes 2 and 3).
2. Run a command, similar to the following, within the folder
that contains both half-maps.
>> /path/to/sxlocres.py /path/to/halfmap1.mrc /path/to/halfmap2.mrc local_resolution_volume.mrc --cutoff=0.143 --fsc=resolution.txt
166
Sriram Aiyer et al.