Job: Sharpening Tools

Re-sharpen and adjust the B-factor of a volume after refinement.

Description

CryoSPARC refinements automatically generate a sharpened map the B-factor found by a Guinier plot. However, in some cases these automatically generated maps may not be best. For example, maps with varying quality may have some regions which benefit from more sharpening and some which require less for accurate model building. Additionally, when comparing maps it is best to compare maps sharpened with the same B-factor. Sharpening Tools allows you to create a sharpened map with a specific B-factor, mask, and filter for these situations.

Input

Input volume

This volume is the map which will be sharpened. The map volume must have both map_half_A and map_half_B (all Refinement jobs produce these outputs).

Mask override

In versions of CryoSPARC before v5, this input was named Input Mask and you either had to provide a mask to this slot or turn on the Generate new FSC mask parameter to create a new FSC mask using the same process as is done in refinements. A job with no input mask and Generate new FSC mask turned off would fail.

Starting with CryoSPARC v5, if this input is left empty, the Input volume's fsc_mask_auto slot will be used instead of failing. If Generate new FSC mask is turned on, the new mask generation procedure will be used.

Output

• A sharpened map

  • map_sharp

• The mask used during the sharpening job

  • mask_fsc

Common Parameters

B-Factor to apply

A negative value to sharpen the map. A general rule of thumb is to start with the B-Factor value presented in the Guinier Plot of a refinement job, e.g., -76.7 for the plot below.

Note that CryoSPARC's Guinier plots report the B-factor as a positive number, but it should be entered here as a negative number.

Numbers closer to 0 will produce a less sharp map, useful if the density looks fragmented. Numbers further from 0 will produce a sharper map, useful for building models into particularly high-quality regions of the map. If your map has regions of higher and lower quality, it is often helpful to load several maps with varying sharpness during model building.

If this parameter is left blank, the B-factor will be estimated using a Guinier plot, as it is during refinement.

Which FSC to filter volume by

New in CryoSPARC v5

After sharpening, volumes are lowpass filtered to remove noise. Three choices of lowpass filter are available:

• full: Filter by the "full" FSC. This is the theoretical FSC between the map and an ideal, noiseless map. This is the default for sharpening, as it is a good estimate of the true quality of the map.

• half: The FSC between the two half maps. This is the FSC used during refinement to limt overfitting; it is generally an underestimate of the resolution of the final, averaged map.

• none: Filter by a Butterworth filter instead of an FSC. This is recommended when comparing several maps such that all maps are filtered and sharpened in the exact same way. See Lowpass filter for more information.

Lowpass filter order and corner resolution

When Which FSC to filter volume by is set to none, a Butterworth filter is used instead of an FSC. As mentioned above, this is generally best when comparing several maps, such that their filtering and sharpening are as close to identical as possible, since even the same mask will produce a different FSC curve if the half maps differ.

Two parameters are necessary to specify a Butterworth filter:

• The Lowpass filter order specified the "steepness" of the filter. A higher order introduces more ringing artifacts, but includes less information beyond the corner resolution. A lower order attenuates ringing, but allow more information beyond the corner resolution.

• The Lowpass filter corner resolution specifies the resolution at which the filter attenuates signal by a factor of one half. Put another way, this is the resolution at which the filter equals 0.5, or is at half-height. When comparing multiple maps, this can be set to the same value for all of them (e.g., the coarsest resolution among all of the maps to compare).

Common Next Steps

Downloading and inspecting the sharpened map in a software such as UCSF Chimera

Download options for the "volume" output group in Sharpening Tools