Job: Align 3D maps
Align two or more 3D maps.
At a Glance
Align any number of input volumes to a reference volume.
Description
When comparing multiple volumes, it is important to have them in the same register. Align 3D Maps accepts a reference volume and any number of input volumes. It will find, for each input volume, the optimal alignment to the reference volume. Typically, each of the volumes would be distinct maps of the same target as the reference — however, Align 3D Maps performs well as long as the maps are similar.
Because Align 3D Maps can also align particles, it is useful for downstream jobs that require input particles to be in-register. Such downstream jobs include refinements without global pose search (e.g. Local Refinement and 3D Flexible Refinement), 3D Classification, and 3D Variability. If combining particles from multiple classes or from multiple refinement jobs as input to any of these jobs, using Align 3D Maps to place all particles and volumes in-register often improves results.
Note that all maps in the input volume are aligned. For instance, the Volume output of a Homogeneous Refinement typically contains the map, the sharp map, and two half maps. If this Volume is provided to Align 3D Maps, all of those contained maps will be aligned.
Inputs
Depending on whether a volumes group (new in CryoSPARC v4.5) or several individual volumes are provided as inputs, the form of other input slots change. The input section of this page is therefore divided into two sections, with some shared information in each.
Inputs when using volumes groups
Reference Map
The input maps will be aligned to this map.
Reference Mask
If this input is provided and Apply mask to volumes is turned on, the reference will be masked before alignment.
Maps to align (volumes group)
This input accepts a single volumes group containing the volumes to be aligned to the reference. If this input is connected, the job will not accept any inputs to the Maps to align (individual volumes) input.
Particles (all)
This input is only visible if Update particle alignments is turned on and an input is connected to Maps to align (volumes group)
Particles in this input will have their 3D alignments updated in agreement with their corresponding map. Note that particle images are not used for alignment.
Masks all classes (individual masks)
This input is only visible if Create mask inputs is turned on and an input is connected to Maps to align (volumes group)
This input accepts a number of masks which will be applied to the input volumes group before alignment, if Apply mask to volumes is turned on.
If exactly one mask is connected to this input, the mask will be applied to all input volumes before alignment. This may be useful if the input volumes are all aligned to each other, but not to the reference (perhaps aligning the results of a 3D Classification job to another refinement). This will almost certainly produce poor results if the input volumes are not aligned to each other.
If more than one mask is connected to this input, then exactly one mask per volume in the volumes group must be connected. For example, if the volumes group contains six volumes, then six individual masks must be added to this input. The masks are applied in order of their addition, i.e., the mask plugged into this input first is applied to the first volume in the group, the mask plugged in second is applied to the second volume in the group, etc.
Inputs when using multiple individual volumes
Reference Map
The input maps will be aligned to this map.
Reference Mask
If this input is provided and Apply mask to volumes is turned on, the reference will be masked before alignment.
Maps to align (individual volumes)
This input accepts one or more individual volumes which will be aligned to the reference.
If this input is connected and a volumes group is subsequently connected to the Maps to align (volumes group) input, the individual volumes will immediately be removed from this input slot.
Particles (map to align, connection N)
These inputs are only visible if Update particle alignments is turned on and inputs are connected to Maps to align (individual volumes)
Particles in this input will have their 3D alignments updated in agreement with their corresponding map. Note that particle images are not used for alignment. A copy of this input is created for each volume connected to Maps to align (individual volumes), with the number N updated to indicate which group the particles will be aligned with.
Note that removing a volume after connecting particles to the inputs does change the other volumes’ numbers but does not change the particle inputs. It is therefore recommended that all volumes are connected before moving on to connect particles.
Mask (map to align, connection N)
These inputs are only visible if Create mask inputs is turned on and if inputs are connected to Maps to align (individual volumes)
The mask plugged into this input will be applied to the indicated volume before alignment, if Apply mask to volumes is turned on. A copy of this input is created for each volume connected to Maps to align (individual volumes), with the number N updated to indicate which group the mask will be applied to and aligned with.
Note that removing a volume after connecting a mask to this input does change the other volumes’ numbers but does not change the mask inputs. It is therefore recommended that all volumes are connected before moving on to connect masks.
Commonly Adjusted Parameters
Update particle alignments
If this parameter is turned on, particle input group(s) will be created as outlined above. Particle poses will be updated to match the new orientation of the volume.
Create mask inputs
If this parameter is on, mask input group(s) will be created as outlined above. If using single volume inputs, masks will be aligned and output along with their corresponding volumes.
Apply mask to volumes
If this parameter is on, masks will be applied to their corresponding volumes before alignment. Note that this controls masking of both the reference and the input volumes — if a mask is connected to the Reference mask input but this parameter is off, the reference will not be masked.
Log rotation matrices, figures, and difference maps
If this parameter is on, the log will contain the rotation and translation matrices and will produce diagnostic plots (see Outputs).
Check both chiralities
If this parameter is on, both chiralities (i.e., both the input version and the “z-flipped” version of the map) of the input maps are checked to find the best alignment in the best hand. If all maps are known to be in the correct hand, this parameter can be turned off to increase the job’s speed.
Maximum shift (A)
Translations only up to this shift are checked. If the maps are significantly out of register, this parameter may need to be increased.
Maximum search angle (degrees)
Rotations only up to this angle are checked. Note that, since angles are checked in all directions, the default value of 180° checks the entire range of possible rotations.
Lowpass filter resolution (A)
Prior to alignment, but after masking, all input maps and the reference map are lowpass filtered to this resolution.
Outputs
Depending on whether a volumes group (new in CryoSPARC v4.5) or several individual volumes are provided as inputs, the output slots change. The output section of this page is therefore divided into two sections, with some shared information in each.
Outputs when using volumes groups
Reference Volume
This output contains the unmodified reference volume.
Aligned volumes
This output contains the aligned input volumes. Note that this is a volumes group output.
All particles
This output contains the input particles with poses updated according to their associated volume.
Outputs when using multiple individual volumes
Reference Volume
This output contains the unmodified reference volume.
Aligned volumes
This output contains the aligned input volumes in a Multi Volume Group.
Aligned volume N
This output contains the Nth input volume, aligned to the reference.
Mask aligned N
This output contains the Nth input mask, aligned in the same pose as the Nth volume. This output only exists if Create mask inputs was enabled.
Particles for map N
This output contains the particles belonging to the Nth volume with updated poses.
Diagnostic Plots
Log messages
Each map reports the rotation matrix and 3D translation used to align it to the reference volume. The log also records whether or not the hand of the input map was flipped.
Difference map
Each aligned volume is subtracted from the reference. Slices through this difference volume are plotted. Note that the difference maps are calculated after alignment and lowpass filtering, so their appearance will change depending on the value selected for the lowpass filter.
This plot is useful in assessing the alignment quality, and highlighting domains which may be hampering a successful alignment. Assuming the two volumes are similar, a well-aligned pair of volumes will have most regions of this plot white (for little to no difference between the input map and the reference).
FSC
The Fourier Shell Correlation between each input volume and the reference. Note that this is a between-volumes FSC, not a GSFSC. Thus, the “resolution” of these plots indicates the frequency up to which the two volumes have a correlation of at least 0.143, not necessarily the reliability of either volume at any given resolution.
Well-aligned volumes will have a relatively high FSC up to the applied lowpass filter frequency applied (or the lower of the two volumes’ GSFSC resolutions).
References
Nguyen, T. H. D. et al. Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Nature 530, 298–302 (2016).
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