Job: Import Particle Stack

At A Glance

Import a stack of particles with metadata and CTF parameters.

Description

The Import Particles job import a stack of extracted particle images (each being a square cropped image of one particle) into CryoSPARC for downstream processing. The particle stack must be in .mrc or .mrcs format, and the metadata for the particles in .star format.

Importantly, the particle stack files that are imported by this job are not copied — they are merely linked (via a symbolic link) into the CryoSPARC project directory where this job is running. It is thus critical that the particle stack files are not deleted or moved while they are being processed in CryoSPARC.

Inputs

Source Exposures (optional)

Some jobs require knowing where in a movie a particle image comes from. For instance, Remove Duplicate Particles requires knowing how close to images are on the movie, and Reference Based Motion Correction corrects motion in each frame of the input movie for each particle. To enable these jobs with imported particles, you must link them to exposures imported separately (e.g., through an Import Movies or Import Micrographs job).

If Source Exposures are provided as input, the job will attempt to link the imported particles to exposures from which they were extract. In this case, the filenames must match those found in the rlnMicrographName of the .star files found in Particle meta path. Parameters to trim prefix and suffixes of the input exposure names and the names found in rlnMicrographName are provided. See Import Micrographs for more information on the use of this type of parameter.

Commonly Adjusted Parameters

Particle meta path

The Particle meta path is required, and contains information about the particles, such as their defocus or pose. This is typically some form of text file, for instance a RELION .star file.

Particle data path

The Particle data path is required, and contains the particle images themselves.

Microscope parameter overrides

One can specify Pixel size (A), Accelerating voltage (kV), Spherical aberration (mm) and Total exposure dose (e/A^2) if known. Otherwise, they will be read from the files found in Particle meta path.

Data Sign determines whether the information in the particle stack is dark particles on a light background or light particles on a dark background. CryoEM data is typically recorded dark-on-light (Data Sign = +1) but can be flipped during processing or extraction in some programs. For instance, RELION flips particles to be light on dark by default during extraction.

CryoSPARC always displays particles with the Data Sign applied; if particles images are stored on disk as light-on-dark and with Data Sign set to -1, the particles will appear dark-on-light.

Outputs

Imported Particles

An imported particle stack for further use in CryoSPARC.

Common Next Steps

Depending on the source of the particles, it may be appropriate to proceed with particle curation (e.g., 2D Classification or Ab-Initio Reconstruction) or other types of refinement (e.g., Non-Uniform Refinement, 3D Flexible Refinement).

Common Problems

Incorrect defocus due to higher-order CTF parameters

RELION and CryoSPARC record higher-order CTF parameters in mutually incompatible formats. Performing higher-order refinements in global CTF in one processing software will require re-fitting CTF in the other.

This is because higher-order effects in the CTF account for some of the aberrations that are otherwise incorporated into the defocus. Since RELION cannot read CryoSPARC’s values for these higher-order effects (and vice-versa), the fitted CTF is now incorrect because it has the modified defocus value but not the higher-order effects. This problem is resolved by re-refining the CTF whenever particles with higher-order aberrations are moved between the two packages.