Local CTF Refinement
adjusts each particle's defocus value to estimate the z-position of the particle in the sample/ice. Global CTF Refinement
adjusts the higher-order CTF terms (beam-tilt, trefoil, spherical aberration, tetrafoil) across an entire group of images to find the optimum values, accounting for misalignment or aberrations in the microscope itself.Global CTF Refinement
now supports the estimation and correction of anisotropic magnification present in the particle images.Local CTF Refinement
in cryoSPARC requires aligned particle images and a 3D reference (two half-maps), ideally already at a high resolution. Experimental particle images are compared against the 3D reference from their half-set, from the best known pose, at various defocus levels, and the best defocus is selected. The optimal defocus ideally corresponds to the height of the particle in the sample/ice.Local CTF Refinement
job using the job builder and connect particles from a previously-run refinement (the particles must have alignments3D
defined). Also connect the refined volume from the same refinement job. You can optionally connect a separate mask input, otherwise the mask_refine
that is included in the volume input from the previous refinement will be used by default.Patch CTF Estimation
previously in cryoSPARC, this value can be made relatively small, about the same as the thickness of ice you expect to have in the sample, since the input defocus values will already be fairly accurate.Global CTF Refinement
generally works best with larger, more rigid particles. However, Global CTF Refinement
does use signal from all the particles in an exposure group, and so can detect beam tilt and other aberrations even with smaller/flexible structures.Global CTF Refinement
job using the job builder and connect particles from a previously-run refinement (the particles must have alignments3D
defined). Also connect the refined volume from the same refinement job. You can optionally connect a separate mask input, otherwise the mask_refine
that is included in the volume input from the previous refinement will be used by default.Global CTF Refinement
job is run, meaning that the plots will always show aberrations in the measured data (first plot). Even terms, on the other hand, are optimized starting from their current input values. Therefore if Global CTF Refinement
is run twice, the second time, the even terms will show nearly zero aberration in the measured data (since the input CTF parameters are already nearly correct).Global CTF refinement
the units of each aberration parameter are printed. Beam-tilt is internally parameterized in Angstroms rather than radians, as converting to the latter requires a non-zero spherical aberration coefficient. Values in milli-radians are printed in cases where the spherical aberration is non-zero.Account for EWS curvature
parameter and make sure to set the EWS curvature sign
to the correct value of curvature determined from previous reconstructions with Ewald Sphere enabled. For more information on how to obtain these reconstructions and the curvature sign, please refer to the Ewald Sphere Correction tutorial for a detailed walkthrough. Local CTF Refinement
and Global CTF Refinement
can be run as standalone jobs. However, since the refinement of these parameters is very fast, they can also be run on-the-fly during iterations of Homogeneous Refinement
. In the new Homogeneous Refinement
job in v2.12+, there are new parameters to enable local and/or global CTF refinement. CTF refinement is carried out iteratively with refinement of 3D poses and the 3D map, starting once the initial refinement is converged.Homogeneous Refinement
job in v2.12+ will create plots similar to the standalone CTF refinement jobs, and the final CTF parameters after refinement will be outputted along with the 3D alignments of particles.Non-uniform Refinement
job has been updated to use the new GPU code that supports higher-order CTF correction, but this is NOT enabled by default. You must turn on the Enable higher-order CTF
parameter in Non-uniform refinement
. Please also note that legacy refinement jobs will not support the correction of high-order CTF aberrations or anisotropic magnification.Non-uniform Refinement
, so particles should be processed through the standalone Local CTF Refinement
then Global CTF Refinement
jobs first.Override Exposure Group ID
parameter. Using this method, you can import your datasets separately based on their beam tilt groups, or any other groups where you would like to use, and the grouping of imports will be retained even if the datasets are merged later on in processing.Exposure Groups
utilitiesExposure Group Utilities
Job. This job allows you to view, split, and combine datasets into one or more exposure groups.Exposure Group Utilities
job, and select the split
mode. Use the parameters Field to use to split Dataset
, Start Slice Index
, and Number of characters to Consider
(more information here) to create unique tokens out of the file paths available. The job automatically creates and sets exposure groups for these tokens:Split Outputs by Exposure Group
parameter.combine&set
mode and Set Exposure Group Value
parameter, you can combine all connected datasets and set their exposure group to the same value. Note that when this happens, the job will check that the CTF values across the exposure group are consistent- you can decide what the job will do if it finds inconsistent values using the Combine Strategy
parameter.ctf/exp_group_id
(and mscope_params/exp_group_id
for movie/micrograph datasets or location/exp_group_id
for particle datasets) for all items inside the dataset. You can set these columns with the desired group identifiers, which do not need to be sequential but do need to be unique.