12. For accurate global FSC calculations, the experimental map
should be masked to remove background noise from the solvent. Many packages can create masks (see also Notes 2 and 3);
we performed this step using the relion_mask_create command
implemented within the Relion software package. For example,
the command used for the AAV2 dataset was:
>> relion_mask_create --i experimental_map.mrc --o output.mrc -ini_threshold 0.001 --width_soft_edge 6 --extend_inimask 6
13. A common mistake is to upload volume files in a format that is
not mrc (e.g., hdf, spi, vol). The current version of the program
only supports mrc format.
14. Other outputs associated with the run are provided for convenience, such as a thresholded and binarized 3D FSC volume, as
well as supplementary curves corresponding to the X, Y, and Z
resolutions relative to the internal coordinate system of the
map (see Note 15 for a word of caution on the lack of a relation
between the internal coordinate system of the map and the
direction relative to the electron beam). The 3D FSC is used to
evaluate the directional resolution of the reconstructed map.
15. In Chimera, the X/Y plane corresponds to the image that is
displayed on the screen, whereas the Z direction is out-of-plane
and is not evident to the eye. The experimental map and the
3D FSC volume are loaded in the first window. The second
window shows the dynamic plot that reflects the FSC for a
given Z-direction (which changes as the map is rotated), as well
as a fixed global FSC curve (see Fig. 4). Due to the geometry of
the imaging experiment, and the fact that particles adhere
along a plane perpendicular to the electron beam, the
Z-resolution relative to the electron beam is typically the worst
resolved direction. However, a word of caution here: the
Z-direction relative to the electron beam does not necessarily
correspond to the Z-direction of the internal coordinate system of the map. The former can be precisely identified, e.g.,
using the tilt-pair test and with knowledge of the tilt angle and
tilt axis of the microscope [10]. The latter inherently depends
on how the orientation angles are assigned to the map. In
practice, the Z-direction relative to the electron beam can
correspond to any direction when the map is initially opened
in Chimera. Due to the geometry of the imaging experiment,
and the fact that particles adhere to the air–water interface
perpendicular to the electron beam, the Z-direction relative
to the electron beam can often be identified by locating the
direction of worst Z-resolution within the second window (see
Note 16).
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should be masked to remove background noise from the solvent. Many packages can create masks (see also Notes 2 and 3);
we performed this step using the relion_mask_create command
implemented within the Relion software package. For example,
the command used for the AAV2 dataset was:
>> relion_mask_create --i experimental_map.mrc --o output.mrc -ini_threshold 0.001 --width_soft_edge 6 --extend_inimask 6
13. A common mistake is to upload volume files in a format that is
not mrc (e.g., hdf, spi, vol). The current version of the program
only supports mrc format.
14. Other outputs associated with the run are provided for convenience, such as a thresholded and binarized 3D FSC volume, as
well as supplementary curves corresponding to the X, Y, and Z
resolutions relative to the internal coordinate system of the
map (see Note 15 for a word of caution on the lack of a relation
between the internal coordinate system of the map and the
direction relative to the electron beam). The 3D FSC is used to
evaluate the directional resolution of the reconstructed map.
15. In Chimera, the X/Y plane corresponds to the image that is
displayed on the screen, whereas the Z direction is out-of-plane
and is not evident to the eye. The experimental map and the
3D FSC volume are loaded in the first window. The second
window shows the dynamic plot that reflects the FSC for a
given Z-direction (which changes as the map is rotated), as well
as a fixed global FSC curve (see Fig. 4). Due to the geometry of
the imaging experiment, and the fact that particles adhere
along a plane perpendicular to the electron beam, the
Z-resolution relative to the electron beam is typically the worst
resolved direction. However, a word of caution here: the
Z-direction relative to the electron beam does not necessarily
correspond to the Z-direction of the internal coordinate system of the map. The former can be precisely identified, e.g.,
using the tilt-pair test and with knowledge of the tilt angle and
tilt axis of the microscope [10]. The latter inherently depends
on how the orientation angles are assigned to the map. In
practice, the Z-direction relative to the electron beam can
correspond to any direction when the map is initially opened
in Chimera. Due to the geometry of the imaging experiment,
and the fact that particles adhere to the air–water interface
perpendicular to the electron beam, the Z-direction relative
to the electron beam can often be identified by locating the
direction of worst Z-resolution within the second window (see
Note 16).
182
Sriram Aiyer et al.