blob, ctf, alignments3D)map, map_half_A, and map_half_B)Threshold and Soft padding width parameters. We typically recommend setting the Soft padding widthparameter to be a voxel value that ensures the resulting padding is several factors larger than the expected resolution of the complex. A good rule of thumb gives the minimum mask softness as 5 * res_angstroms / pixel_size_angstroms.For example, a complex reaching 3.0Â Ã… in resolution with a pixel size of 1.0 Ã… should be padded to at least 5 * 3.0 / 1.0 = 15 voxels. Use pose/shift gaussian prior during alignment parameter, and by specifying theStandard deviation of prior over rotation/shift parameters. Rotation search extent (deg): This controls the rotational extent, in degrees, to which particles are allowed to deviate from their initial poses.Shift search extent (A): This controls the translational extent, in pixels, to which particles are allowed to deviate from their initial shifts.Standard deviation (deg) of prior over rotation: This can be set to a number in degrees, corresponding to the desired standard deviation of an isotropic Gaussian prior over rotation. This has the effect of softly penalizing poses that are far away from the initial poses, rather than strictly cutting off the search beyond a specific point. Note that by default, the rotational search extent will be set to three times this value, but both parameters can be independently specified if desired.Standard deviation (A) of prior over shifts: This can be set to a number in Angstroms, corresponding to the desired standard deviation of an isotropic Gaussian prior over shifts. This behaves similarly to the Gaussian prior over rotation. Note that the "Use pose/shift gaussian prior during alignment" parameter must be set to true to activate both priors.Default fulcrum location: Local Refinement uses a default fulcrum centered at the center of mass of the mask input to the job. This can be changed to use the center of the box (i.e. the rotation center used by all other refinements). Optionally, the fulcrum can be manually overwritten by typing comma-separated coordinate values in the "Override fulcrum coordinates (pix)" parameter, which corresponds to the grid index of the desired fulcrum in x, y, z order, indexed relative to the corner of the box. Re-center rotations/shifts each iteration?: Local Refinement allows for the pose and shift search grids to drift between each iteration, so that the alignment search grids can be re-centered on the previous iteration's optimal pose/shift. If these parameters are activated, it is recommended to also activate the gaussian prior, as poses and shifts may stray too far in early iterations from the true optima. Note that when re-centering is active, both the rotation/shift search grids as well as the rotation/shift gaussian priors will be re-centered at each iteration.Maximum alignment resolution (degrees): This specifies the finest alignment search grid spacing to use, which will be enforced by the final branch-and-bound iterationMarginalization: This enables efficient pose and shift marginalization over particle alignments during reconstruction steps. Marginalization can improve 3D structure quality on smaller proteins, decrease overfitting, and sometimes limit the influence of outlier images.Non-Uniform Refinement Enable: Non-Uniform Refinement can be enabled to help resolve molecules with spatially-varying local resolution. This is particularly useful for membrane proteins, or any proteins with disordered regions (e.g. micelles).Non-uniform filter order: This controls the order of the butterworth filter used for cross-validation-optimal regularization.Non-uniform AWF: Adaptive Window Factor for cross-validation-optimal regularization. Trade off between fast transitions between regions (AWF should be lower) and more accurate local cross-validation test (AWF should be higher).Symmetry: Local refinement supports enforced point-group symmetry around the object origin (i.e. the center of box, not the fulcrum). Note that this parameter, should not be changed if the particles have previously been symmetry expanded. For most use cases, symmetry expansion is still preferred over enfirced symmetry, as it incorporates symmetry while also allowing particle alignments between each asymmetric unit to vary slightly, which can account for flexibility between asymmetric units.Mask (dynamic, static): This can be set to dynamic to re-generate a dynamic mask at each iteration of the refinement, or instead to static to use the unmodified input mask at each iteration. Note that we generally recommend static masks to be use, as we have seen that dynamic masks can result in mask-based overfitting.Dynamic mask threshold (0-1): The level-set threshold for selecting regions to include in the dynamic maskDynamic mask near (A): The distance to dilate the dynamic mask to, after thresholdingDynamic mask far (A): The distance at which the mask becomes 0.0Minimize over per-particle scale: This allows the per-particle scale factor to be optimized at each refinement iteration. For most use cases of Local Refinement, this should be kept off, as the comparison between a masked subregion and each particle image may not be accurate if the mask excludes a large portion of the volume.