Tool: Segment Map

Segment Map partitions volume data to create a surface model with one or more segmentation regions shown in different colors. Along with the Fit to Segments tool, Segment Map is part of the Segger package described in:

Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. Pintilie GD, Zhang J, Goddard TD, Chiu W, Gossard DC. J Struct Biol. 2010 Jun;170(3):427-38.

Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps. Pintilie G, Chiu W. Biopolymers. 2012 Sep;97(9):742-60.

Segmentation regions can be measured with Measure and Color Blobs and saved as an index map with segger exportmask.

For interactive (manual) segmentation of medical images, see the Segmentations tool. See also: Volume Viewer, Map Eraser, Map Filter, color zone, volume mask, segmentation, EMDB segmentation file format (SFF), Segger website, Chimera-Segger how-to (2010)

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Methodology

Segment Map performs watershed segmentation: a density map is partitioned so that each local maximum has its own region, and the boundaries between regions lie at the valleys between the local maxima. The goal is usually one region per protein or domain, but the initial segmentation often gives more numerous and smaller regions than desired, especially if the data are noisy. Segger provides various ways to combine regions after the initial segmentation:

• automatic grouping by scale-space filtering (smoothing the data), done by default; however, smoothing the data before segmentation may be necessary, as discussed below
• interactive grouping by manual selection
• interactive grouping by connectivity
• interactive grouping using fitted atomic models

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Preprocessing

It may be necessary to process a density map beforehand for the segmentation to finish in a reasonable amount of time. A map of size 256³ can have a million local maxima if it is noisy. Gaussian smoothing to reduce the number of local maxima can be performed with the command volume gaussian. Gaussian filtering reduces resolution to about six times the Gaussian standard deviation. After smoothing, it will also improve performance to reduce the size of the map with volume bin. For visualization and analysis, it is desirable for a density map to be oversampled by three times in each dimension, so binning should aim for a grid spacing of approximately twice the Gaussian standard deviation.

If interactive rotation speed is poor after segmentation, see response time.

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Dialog and Basic Usage

Segment Map can be started from the Volume Data section of the Tools menu (more...).

The map of interest should be chosen from the pulldown menu of open volume data sets next to Segment map. The threshold (contour level) of the map should be adjusted before segmentation, because the calculation will use only the voxels with density values above that level.

Clicking Segment partitions the chosen map by the watershed method plus smoothing and grouping as specified in the segmenting options. The segmentation result is shown as a surface model with one or more segmentation regions of different colors. The resulting number of regions is reported in the dialog and the Log. The segmentation is given the same name as the corresponding map except with .seg appended.

The Current segmentation is indicated in the dialog and can be changed by choosing a different segmentation name from the pulldown menu. Designating a segmentation as the current segmentation shows it and hides any others. Grouping and ungrouping operations modify the current segmentation model rather than creating a new one. However, multiple segmentation models can be open at the same time, and may have been generated from different density maps or opened from previously saved segmentation files.

Grouping and ungrouping can be specified interactively by selection. With default mouse-button assignments, Ctrl-click selects a region and Shift-Ctrl-click selects additional regions without erasing the pre-existing selection.

• If no segmentation regions are selected, clicking Ungroup backtracks to the previous stage of grouping of the current segmentation. By default, Segment automatically applies multiple rounds of smoothing and grouping, so that different intermediate stages can be accessed by clicking Ungroup multiple times. If regions are selected, Ungroup will backtrack to the previous stage of grouping for the selected region(s).
• If no segmentation regions are selected, Group traverses the stages in the opposite direction as Ungroup, or performs further grouping. If regions are selected, Group will combine the selected regions into a single group.

Segmentation region selection, showing/hiding, and other manipulations can also be performed with the shortcuts options or the Segment Map Regions menu.

Segmentation results are included in saved sessions. They can also be saved separately using the Segment Map File menu or the save command.

Clicking Fit opens the Fit to Segments tool. Clicking Help shows this page in the Help Viewer.

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Segmenting Options:

• Display at most [maxreg] regions, granularity [triangle-size] voxels – limit the number of displayed regions to maxreg (default 60 largest) and set surface triangle-size (default 1). If more regions than maxreg are generated, however, those which are not displayed will still be included in subsequent smoothing and grouping calculations. Smaller surface triangles give a smoother appearance, but more triangles take longer to render. The default triangle-size of 1 gives triangles comparable in size to the map grid spacing. A value of 2 will make the triangles twice as large (quadruple the area) and reduce their number by about a factor of 4. If response time after segmentation is slow, try decreasing maxreg and/or increasing triangle-size.
  
  
• Keep regions having at least [minsize] voxels, [mincontact] contact voxels – throw out regions containing fewer than minsize voxels (default 1) or having fewer than mincontact contact voxels (default 0) with other regions. These apply only to the initial segmentation, not after smoothing. The default settings retain all regions.
  
  
• Group by smoothing [N] steps of size [M], stop at [minreg] regions – whether to group by smoothing right after watershed segmentation (when the Segment button is clicked), and if so, how many rounds of automatic smoothing and grouping to perform (default N=4) and how much smoothing to perform at each round (default M=1). If a round would give fewer than minreg regions (default 1), the process is backtracked to the previous round of automatic grouping. Grouping by smoothing is on by default and mutually exclusive with grouping by connectivity.

  If L is the minimum of the grid spacing along the three axes, the standard deviation of the Gaussian used for smoothing will be MxL in the first round and increase by MxL in each subsequent round. A single round consists of smoothing the data, then letting each local maximum point from the previous round move by steepest ascent to the location of a new local maximum. If two or more previous maxima converge on a new maximum, their regions from the previous round are coalesced into a single region. In general, the number of rounds N is the dominant parameter to adjust when fewer, larger regions are desired. A larger step size M may be beneficial for noisier maps. However, a smaller step size gives more gradual changes per round, thus more rounds to achieve a similar end result, and more intermediate stages that can be traversed with the Group and Ungroup buttons. After the automatic rounds have completed, clicking Group executes the minimum number of additional rounds needed to change the grouping further.

• Group by connectivity [N] steps, stop at [minreg] regions  ☐ only visible – whether to group watershed regions based on density connections, and if so, whether to use only the visible regions. If the density map has not been segmented already, watershed segmentation will use only the voxels with density values above the current threshold without subsequent smoothing/grouping. Clicking Group will then perform the specified number of rounds (steps) of grouping by connectivity (default N=20). Each round corresponds to one pass through contact list. If a round would give fewer than minreg regions (default 1), the process is backtracked to the previous round. If grouping has already been performed, some ungrouping may be needed before using the connectivity method, as it will not group watershed regions together if they are already in different groups.

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• Select regions:
  • All
  • Over Sel. Atoms – those overlapping selected atoms
  • Invert – invert selection
  • Not-Grp – those not containing any subregions
  • Grouped – those comprising ≥ 2 subregions
• Selected regions: (act on selected regions)
  • Hide
  • Show
  • Delete
  • Tr. – transparent surface
  • Opaque – opaque surface
  • Mesh
• Show regions:
  • None
  • All – all (but subject to the Display at most... setting)
  • Only Sel. – selected regions
  • Adj. – regions adjacent to any selected regions
  • Not-Grp – those not containing any subregions
  • Grouped – those comprising ≥ 2 subregions
• Other tools:
  • Fit – Fit to Segments
    – not implemented –
  • Extract – detailed options for extracting densities within the selected regions
  • iSeg – radial/icosahedral segmentation
  • ProMod – probabilistic modeling of multiple conformations
  • ModelZ – assess how well structural features are resolved

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Segment Map Menu

Menu entries apply only to the current segmentation unless stated otherwise.

• Open segmentation... open a previously saved Segger segmentation file
• Save segmentation – save the segmentation to a Segger file, name/location previously specified
• Save segmentation as... save the segmentation to a Segger file, specify name/location
• Save selected regions to .mrc file... save density map masked by the selected segmentation regions to an MRC file (map dimensions set to the minimal box containing the regions)
• Save all regions to .mrc file... save density map masked by all segmentation regions collectively as an MRC file (map dimensions set to the minimal box containing the regions)
• Save each region to .mrc file... save density map masked by each selected segmentation region as a separate MRC file (map dimensions set to the minimal box containing the corresponding region); if no regions are selected, all are used
• Close segmentation – close the current segmentation model
• Close all segmentations except displayed – close all hidden segmentation models
• Close all segmentations – close all segmentation models
• Associate Selected – associate the current segmentation with the map currently chosen in the dialog (useful when the map has been opened separately or from a different location than expected by the session with the segmentation results)

• Show all – show all segmentation regions at the current level of grouping
• Show only selected – show the selected segmentation regions, hide all others
• Show adjacent – show regions in contact with the selected regions
• Show grouping – show the watershed regions within groups rather than the larger group surfaces; affects the selected regions, or if none are selected, all regions; does not change the groupings, only how they are shown
• Unshow grouping – show the larger group surfaces rather than the watershed regions; affects the selected regions, or if none are selected, all regions
• Hide – undisplay the selected regions, or if none are selected, all regions
• Make transparent – make 55% transparent the selected regions, or if none are selected, all regions
• Make opaque – make opaque (0% transparent) the selected regions, or if none are selected, all regions
• Color density map – color the density map display to match all regions (subsequently changing the threshold will erase the coloring for volume displays in the surface or mesh style)
• Select groups – select all regions composed of grouped watershed regions
• Select boundary regions – select regions that contain at least one grid point within three voxels of the edge of the density map
• Invert selection – deselect the selected regions and vice versa
• Regions overlapping current selection – select any regions that overlap well with the currently selected atoms, deselect the atoms
• Delete selected regions – delete the selected regions (cannot be undone)
• Delete all except selected – delete all regions except those which are selected (cannot be undone)
• Enclosed volume – report the approximate total volume enclosed by the selected regions (number of grid points and ų value obtained by multiplying the number of grid points by the voxel volume); more precise measurements can be obtained with Measure and Color Blobs
• Mean and SD – report density map mean and standard deviation within each selected region of the current segmentation, which could be based on a binned version of the chosen map
• Extract densities... not yet implemented
• Subtract selected from map – inverse-mask the density map with the selected regions (create a new map: set the density values inside the regions to zero, copy values elsewhere, maintain original map dimensions)
• Show axes for selected – show principal axes of inertia as arrows for each selected region
• Hide all axes – remove all principal axes arrows
• Attributes table... not yet implemented
• How many sub-regions – report number of subregions that have been grouped into the selected region(s)

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Response Time

High number of surface triangles. The number of surface triangles can be reduced by increasing the granularity (triangle size). Doubling the value will reduce the number of triangles by approximately a factor of 4 and may improve rendering speed by a similar factor. The display can also be limited to a specified maximum number of regions.

Selected surfaces. ChimeraX shows selections with outlines, which increase the time to draw a frame. Clearing a selection when it is not needed improves performance.