xmipp3.protocols.protocol_resolution3d module
- class xmipp3.protocols.protocol_resolution3d.XmippProtResolution3D(**kwargs)[source]
Bases:
ProtAnalysis3DComputes the resolution of 3D volumes using the Fourier Shell Correlation (FSC) criteria. The protocol requires two volumes, which are assumed to be independently reconstructed. In addition, the protocol can also compute the B-factor for the volumes.
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## Overview
The Resolution 3D protocol estimates the resolution of a 3D reconstruction by computing Fourier Shell Correlation, or FSC, between two independent maps.
FSC is one of the standard criteria used in cryo-EM to assess the agreement between two 3D volumes as a function of spatial frequency. If two independently derived maps agree at high spatial frequencies, this suggests that the corresponding structural features are reproducible. If the FSC drops at lower frequencies, the map is supported only at coarser resolution.
This protocol can compute FSC in two ways. It can compare an input volume with a separate reference volume, or it can use the two half maps associated with the input volume. The half-map mode is commonly used for resolution assessment because half maps are independently reconstructed from two halves of the data.
The protocol can also compute structure-factor information used for estimating a B-factor, which can later be used for map sharpening.
## Inputs and General Workflow
The protocol requires an input volume.
If FSC calculation is enabled, the protocol needs either:
two half maps associated with the input volume; or
a separate reference volume.
The protocol then runs the Xmipp FSC calculation, producing an FSC metadata file. In addition to FSC, the calculation also includes DPR information.
If B-factor computation is enabled, the protocol computes the volume structure factor. The resulting file can be used by the analysis tools to estimate or apply a B-factor.
Finally, the protocol creates an output FSC object that can be viewed and analyzed in Scipion.
## Volume to Compare
The Volume to compare parameter is the main input volume.
This is the map whose resolution or agreement with another map will be evaluated. It may be a reconstruction, a post-processed map, or a volume produced by another Scipion protocol.
The input volume must have a correct sampling rate, because spatial frequencies are converted into resolution values in angstroms.
If the half-map mode is used, this volume must also contain links to its associated half maps.
## Calculate FSC and DPR
The Calculate FSC and DPR? option controls whether the protocol computes the Fourier Shell Correlation and Differential Phase Residual information.
When this option is enabled, the protocol compares the two selected maps in Fourier space and writes an FSC metadata file.
The summary reports resolution estimates at commonly used thresholds, including:
FSC = 0.5;
FSC = 0.143;
DPR = 45 degrees.
These values provide numerical reference points for interpreting the map agreement.
## Use Half Maps
The Use half maps option tells the protocol to compute the FSC between the two half maps associated with the input volume.
This is usually the preferred mode when half maps are available, because the two maps are expected to be independently reconstructed from separate halves of the particle data.
The protocol checks that the input volume actually has half-map information. If no half maps are associated with the input volume, the protocol reports a validation error.
Half-map FSC is useful for estimating the reproducible resolution of a reconstruction.
## Reference Volume
The Reference volume parameter is used when half maps are not used.
In this mode, the protocol computes the FSC between the input volume and the selected reference volume.
This comparison is useful when the user wants to compare two independent reconstructions, two processing results, or a map against a reference map. However, the interpretation depends on how the two volumes were generated. If the maps are not independent, the FSC may overestimate reproducible information.
The reference volume should have the same box size, sampling, orientation, and position as the input volume for the FSC to be meaningful.
## FSC Curve
The FSC curve describes correlation between the two maps as a function of spatial frequency.
At low spatial frequencies, FSC usually reflects agreement in the overall shape of the map. At high spatial frequencies, it reflects agreement in finer details.
A curve that remains high until high spatial frequencies indicates stronger agreement at finer resolution. A curve that drops early indicates that the maps agree only at lower resolution.
The protocol stores the FSC curve in an output FSC object, which can be plotted and inspected in Scipion.
## FSC Thresholds
The protocol reports resolution values at two FSC thresholds.
The FSC = 0.5 criterion is a traditional, stricter threshold. It reports the resolution at which the FSC curve falls below 0.5.
The FSC = 0.143 criterion is widely used in modern cryo-EM half-map validation. It gives a less strict but commonly reported resolution estimate.
The protocol estimates the crossing point by interpolation between neighboring frequency samples. If the curve does not cross the threshold, the reported resolution may be unavailable.
## DPR Criterion
The protocol also reports a DPR = 45 degrees resolution estimate.
DPR stands for Differential Phase Residual. It measures phase agreement between the two volumes in Fourier space. A smaller DPR indicates better phase agreement.
The reported DPR resolution corresponds to the point where the DPR reaches 45 degrees.
This value complements FSC and provides another way to summarize agreement between the two maps.
## Calculate B-Factor
The Calculate B-factor? option computes structure-factor information from the input volume.
The B-factor describes the falloff of signal amplitude with spatial frequency. In cryo-EM, an estimated B-factor can be used to guide sharpening, where higher-resolution features are enhanced to compensate for attenuation caused by the microscope, detector, reconstruction, and other effects.
This implementation follows an automated approach based on the methodology of Rosenthal and Henderson.
After the protocol finishes, the B-factor can be applied through the analysis GUI.
## Structure Factor File
When B-factor computation is enabled, the protocol writes a structure-factor metadata file.
This file contains the information used to estimate the B-factor. It is not a map by itself, but a diagnostic and post-processing support file.
The protocol summary reports the structure-factor file, and when the B-factor is available, it also reports the estimated value.
## Output FSC
The main output is outputFSC, created when the FSC file exists.
This output stores the FSC curve as a Scipion FSC object. It can be visualized and used for resolution assessment.
The output FSC is linked to the input volume and, when a separate reference volume is used, also to the reference volume.
If only B-factor computation is requested and FSC is disabled, the main result is the structure-factor information rather than an output FSC object.
## Interpreting the Results
The FSC resolution values should be interpreted as indicators of map agreement, not as complete proof of biological correctness.
A high-resolution FSC estimate is meaningful only when the two maps are independent or appropriately separated, properly aligned, and not affected by masking or overfitting artifacts.
Half-map FSC is commonly used because the half maps are reconstructed from separate subsets of particles. Comparison with an external reference can be useful, but its interpretation depends on the independence and similarity of the two maps.
B-factor estimation is useful for sharpening, but excessive sharpening can amplify noise. The sharpened map should always be inspected together with the original map and validation information.
## Practical Recommendations
Use half maps when available. This is usually the most appropriate way to estimate the resolution of a reconstruction.
Use a separate reference volume when you specifically want to compare two maps. Make sure that they are aligned, have the same box size, and represent the same structure.
Report both FSC = 0.143 and FSC = 0.5 values when useful, but inspect the full FSC curve rather than relying only on a single number.
Use B-factor estimation as a guide for sharpening, not as an automatic guarantee of improved interpretability.
Be cautious when comparing post-processed or heavily masked maps, because masking and post-processing can affect FSC behavior.
## Final Perspective
Resolution 3D is a validation and map-assessment protocol.
For biological users, its main role is to quantify how reproducible a 3D map is in Fourier space, either by comparing two half maps or by comparing an input map with a reference volume.
The protocol provides FSC and DPR resolution estimates, and optionally structure-factor information for B-factor estimation. These results should be used together with visual inspection, local resolution, map-model fit, and biological plausibility when assessing a cryo-EM reconstruction.