xmipp3.protocols.protocol_random_conical_tilt module
- class xmipp3.protocols.protocol_random_conical_tilt.XmippProtRCT(**args)[source]
Bases:
ProtInitialVolumeCreates initial 3D volumes using 2D classes and particle pairs from tilted images. Applies the Random Conical Tilt (RCT) method to generate unbiased starting volume for structure refinement, even with a small number of image pairs. The volume serves as starting models for further refinement steps.
AI Generated
## Overview
The Random Conical Tilt protocol creates one or more initial 3D volumes from tilted-pair particle data using the Random Conical Tilt, or RCT, method.
RCT is a classical strategy for obtaining initial 3D models from pairs of untilted and tilted images. The untilted particles are first aligned or grouped into 2D classes. The corresponding tilted particles are then used, together with the known tilt geometry, to reconstruct a 3D volume.
This protocol is useful when the user has acquired tilted-pair data and wants to generate starting models for later 3D refinement. Because the geometry of the tilted acquisition provides important orientation information, RCT can produce initial volumes even when no previous 3D reference is available.
The output is a set of reconstructed initial volumes. Optionally, the protocol also produces low-pass-filtered versions of those volumes.
## Inputs and General Workflow
The protocol requires two related inputs:
a set of tilted-pair particles;
a set of input particles or 2D classes with alignment information.
The tilted-pair particle set provides the relationship between each untilted particle and its tilted counterpart, together with the tilt-angle information. The input particles or classes define the aligned untilted views that will be used to organize the reconstruction.
The protocol first creates Xmipp metadata linking each untilted particle, tilted particle, 2D alignment, micrograph pair, and tilt angle. Then, for each input class or particle group, it aligns the tilted particles with respect to the corresponding untilted reference and reconstructs a 3D volume using ART reconstruction.
If filtering is enabled, each reconstructed volume is also low-pass filtered.
## Input Particles Tilt Pair
The Input particles tilt pair parameter should point to a ParticlesTiltPair object.
This object contains two linked particle sets:
the untilted particles;
the tilted particles.
It also contains the coordinate-pair and micrograph-pair information needed to associate each particle with its corresponding tilted view. The tilt angles are read from the paired-coordinate metadata.
This input is the geometrical foundation of the protocol. If the tilted and untilted particles are not correctly paired, the reconstructed RCT volumes will not be reliable.
## Input Classes or Particles
The Input classes parameter can be either a set of particles or a set of 2D classes.
These input images must contain 2D alignment information. The protocol checks this requirement before running.
If a SetOfParticles is provided, the protocol reconstructs one RCT volume from that particle set.
If a SetOfClasses is provided, the protocol reconstructs one RCT volume for each class. This is a common use case: untilted particles are first classified into 2D classes, and then each class is used to generate a separate initial 3D volume from the corresponding tilted particles.
Using classes can help separate different views, conformations, or particle subsets before RCT reconstruction.
## 2D Alignment Requirement
The input particles or classes must have alignment information.
This alignment describes how the untilted particles are positioned and rotated within the 2D class or particle set. The protocol uses this information when creating the RCT metadata and when relating untilted and tilted images.
If the input particles or classes are not aligned, the RCT reconstruction will not have a meaningful orientation framework.
In practice, users should usually perform 2D alignment or 2D classification on the untilted particles before running this protocol.
## Creating RCT Metadata
The protocol creates an Xmipp metadata file containing the information needed for RCT reconstruction.
For each particle, the metadata includes:
the untilted particle image;
the tilted particle image;
the untilted and tilted micrograph names;
the particle coordinates;
the 2D alignment parameters;
the tilt-pair angles;
the particle identifier.
This metadata is created separately for each input class or particle group.
The protocol also accounts for possible differences in sampling rate between the tilted-pair particles and the input aligned particles by scaling the 2D alignment parameters accordingly.
## Thin Object Option
The Thin Object option allows the protocol to stretch tilted projections to better match the untilted projections.
This option may be useful when the specimen is physically thin and the effect of tilting can be approximated by a stretching operation in projection.
It should be used only when this assumption is appropriate for the specimen. For globular or thick particles, enabling this option may introduce an inappropriate deformation.
## Maximum Allowed Shift for Tilted Particles
The Maximum allowed shift for tilted particles parameter defines the largest allowed shift, in pixels, during alignment of the tilted particles.
Tilted particles whose estimated shift exceeds this value are discarded.
This is a quality-control parameter. Very large shifts may indicate incorrect pairing, poor tilted-image quality, wrong alignment, or particles that cannot be reliably matched between tilted and untilted views.
A larger value is more permissive and discards fewer particles. A smaller value is stricter and may remove problematic tilted particles, but it may also remove valid particles if the initial alignment is difficult.
If the value is set larger than the image size, effectively no particles are discarded by this criterion.
## Skip Tilted Translation Alignment
The Skip tilted translation alignment option disables translation alignment of the tilted particles.
Normally, the protocol aligns the tilted images with respect to the untilted reference or class average. This helps improve the consistency of the tilted particles before reconstruction.
However, if the tilted images have very low quality, translation alignment may produce poor estimates and may make the reconstruction worse. In such cases, skipping tilted translation alignment can be safer.
This is an advanced option and should be used when the user has reason to suspect that tilted-particle translation alignment is unreliable.
## Reconstruction Parameters
The Additional reconstruction parameters field allows the user to provide extra options for the Xmipp ART reconstruction program.
The default parameters are intended as a reasonable starting point for RCT initial-volume reconstruction.
Advanced users can modify these parameters to change reconstruction behavior, such as the number of iterations or regularization-related settings. However, these options should be changed carefully because RCT reconstructions are often based on limited and noisy tilted-pair data.
Most users should keep the default reconstruction parameters unless they have a specific reason to tune the ART reconstruction.
## Filtering Reconstructed Volumes
The Filter reconstructed volumes? option applies a low-pass filter to the reconstructed RCT volumes.
RCT volumes are often noisy, especially when they are reconstructed from a small number of particle pairs or from tilted images with low signal-to-noise ratio. Low-pass filtering can make the initial volume easier to interpret and more suitable as a starting reference.
The protocol can output both the unfiltered reconstructed volumes and the filtered versions.
## Resolution of the Low-Pass Filter
The Resolution of the low-pass filter parameter defines the cutoff used for filtering the reconstructed volumes, expressed in digital frequency.
Lower cutoff values apply stronger low-pass filtering. Higher values preserve more high-frequency information.
For initial models, moderate low-pass filtering is usually appropriate because the goal is to obtain the correct overall shape and orientation, not to interpret high-resolution features.
Excessively weak filtering may leave noisy artifacts in the initial volume, whereas excessively strong filtering may remove useful shape information.
## Output Volumes
The main output is outputVolumes.
This output contains one reconstructed RCT volume for each input particle group or 2D class. If the input is a single particle set, one volume is produced. If the input is a set of classes, one volume is produced per class.
The volumes are converted to MRC format and registered in Scipion with the sampling rate of the untilted particles from the tilted-pair input.
These volumes are intended mainly as initial models for further refinement.
## Output Filtered Volumes
If filtering is enabled, the protocol also produces outputFilteredVolumes.
These are low-pass-filtered versions of the RCT reconstructions. In many workflows, the filtered volumes are more useful as starting references because they suppress noise and emphasize the global shape.
Users should inspect both filtered and unfiltered outputs when deciding which volume to use for downstream refinement.
## Interpreting RCT Volumes
RCT volumes should be interpreted as initial models, not as final high-resolution reconstructions.
They are often affected by missing information, noise, limited angular coverage, tilted-image quality, and the number of particles contributing to each class. Their main value is to provide a plausible 3D starting point for later refinement.
When one volume is reconstructed per 2D class, differences between RCT volumes may reflect different views, conformational states, particle quality, or class composition. Some classes may produce better initial volumes than others.
## Practical Recommendations
Before running RCT, make sure that tilted and untilted particles are correctly paired.
Use 2D classes from untilted particles when possible. Class averages provide cleaner references for aligning the tilted particles than individual noisy particles.
Use classes with enough particles. Very small classes may produce noisy or unstable RCT volumes.
Keep filtering enabled for most initial-model workflows. Filtered RCT volumes are usually more useful for subsequent refinement.
Inspect the tilted-particle alignment and the resulting volumes. Discarded particles or poor volumes may indicate problems in tilted-pair picking, pairing, or class quality.
Use the maximum-shift parameter to reject tilted particles that cannot be aligned reliably, but avoid making it too strict at the beginning.
Treat the output as an initial reference. It should usually be refined further with a standard 3D refinement protocol.
## Final Perspective
Random Conical Tilt is an initial-volume reconstruction protocol for tilted-pair cryo-EM data.
For biological users, its main value is that it can generate a 3D starting model from experimental tilt-pair geometry, without requiring an external reference. This is especially useful for new specimens or workflows where reference bias should be minimized.
The reliability of the output depends strongly on correct tilted-pair coordinates, good untilted 2D alignment or classification, and sufficient tilted-particle quality. Used carefully, RCT provides a practical bridge from paired 2D observations to an initial 3D model.