Models #1 1bzm and #2 1urt associated with sequences in an alignment, with ChimeraX selection sequence region.

Tool: Sequence Viewer

The Sequence Viewer displays individual sequences and sequence alignments of amino acids and nucleotides, with crosstalk to any associated structures. Some of its functions are implemented in the sequence command. To find and select a specified sequence or sequence pattern, use Select... Sequence in the menu or the select command. See also: Modeller Comparative, Model Loops, Coloring by Sequence Conservation

Sequences for biopolymer structures in ChimeraX can be shown:

• with the menu: Tools... Sequence... Show Sequence Viewer and choosing which chain sequence(s) to show
• with the sequence chain command
• by clicking the Molecule Display icon
• by clicking the chain description link that appears in the Log when a structure is opened

Other ways to start the Sequence Viewer: Independent of structure, sequence alignments and individual sequences can also be opened from files or fetched from UniProt. Other tools or commands may generate new sequence alignments (e.g., Blast Protein results, Matchmaker, sequence realignment).

Multiple Sequence Viewer windows can be shown at the same time, and they can be manipulated like other panels in ChimeraX (more...).

Context Menu
Headers and Residue Attributes
Regions
    Region Browser
    UniProt Sequence Features
Sequence-Structure Association
Settings

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Sequence Viewer Context Menu

Each Sequence Viewer window has a context menu with choices including:

• File
  • Save As  ▶ [various sequence file formats] – save to file; see also save
  • Load Sequence Coloring File... open a sequence coloring file to create regions and color associated structure to match
• Edit
  • Copy Sequence... copy a sequence into the system text buffer so that it can be pasted into another application window. Copying options:
    • Copy [sequence name] to system paste buffer
    • Remove gaps from copy (default on) – exclude gap characters (typically present when the sequence is part of a multiple alignment)
    • Restrict copy to active region (default off) – only include residues that are within the active region, even if they form disjoint segments
  • Realign Sequences with Clustal Omega (details...)
    • in new window
    • in this window – replace existing alignment
  • Realign Sequences with MUSCLE (details...)
    • in new window
    • in this window – replace existing alignment
• Structure
  • Associations... show dialog for controlling sequence-structure association
  • Expand Selection to Columns – expand the current selection to all residues associated with the same column(s) of the sequence alignment. See also: sequence expandsel
  • Select by Column Identity – select residues associated with alignment columns that have the specified percentage (100%, <100%, ≥50%, or <50%) of the most prevalent nongap character in that column; see also residue attributes
  • Update Chain Sequence... show dialog for updating structure metadata with the associated sequence(s); this is useful for adding full sequence information to structures read from files lacking PDB SEQRES records or the mmCIF equivalent (details...). For example, the full sequence could be opened from a FASTA file or by fetching it from UniProt, associated with the chain, and then used to update the chain's sequence information. See also: sequence update
  • View [ residue ] – focus the view on the structure residue associated with the clicked sequence position
• Headers – which headers are shown currently
  • Consensus
  • Conservation
  • RMSD – either single atom per residue (Cα for peptides, C4' for nucleic acids) or backbone as per the Headers settings
  • Save  ▶ [choice of available headers] – save header information to file
• Numberings – which sequence numberings to display, if any
  • Overall – whether to show alignment numbering across the top at positions 1, 11, 21, etc.; starts at 1 and includes gaps unless a reference sequence has been designated
  • Reference Sequence – use the individual numbering of one of the sequences:
    • [list of sequences in alignment]
    ...as the overall numbering instead of the default; when the reference sequence has a gap character at a position where a number is to be shown (1, 11, 21, etc.), how that position falls relative to the reference is shown in parentheses. For example, “(<1)” would be shown for alignment positions before the reference's first residue, and “(43/44)” for positions that fall between residues 43 and 44 of the reference.
  • Left Sequence – whether to show individual sequence numberings on the left
  • Right Sequence – whether to show individual sequence numberings on the right
• Tools
  • Modeller Comparative Modeling... start Modeller Comparative (requires a structure to be associated with any sequence in the alignment, for use as a template for comparative modeling)
  • Model Loops... start Model Loops to build missing segments or generate additional conformations of existing segments (requires the structure of interest to be associated with a sequence)
  • Blast Protein  ▶ [if multiple alignment, choice of sequence name] – start Blast Protein with the specified sequence as the query
  • [if multiple alignment] Percent Identity... calculate pairwise percent identities, the number of identical columns between two sequences as a percentage of the specified denominator: length of the shorter sequence (default), or the length of the longer sequence, or the number of columns where neither sequence has a gap. Results are given in the Log. See also: sequence identity
• Sequence Features... for sequences fetched from UniProt, show the features dialog
• Settings... show the Sequence Viewer preferences to control wrapping, spacing, coloring, etc.

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Headers and Residue Attributes

Sequence headers are rows of information above the sequences, such as a Consensus sequence, Conservation histogram, or histogram of the RMSD (root-mean-square displacement) among multiple associated structures. Which headers are shown initially and how they are calculated can be controlled in the Headers settings. Headers for a specific window can be shown, hidden, or saved to a file with the Sequence Viewer context menu or the sequence header command. Numbering can also be controlled using the Sequence Viewer context menu.

Per-column header values are assigned as attributes of the structure residues associated with sequences in the alignment. The numerical attributes (those from headers displayed as histograms) are named “seq_name,“ where name is the name of the header, for example, seq_conservation or seq_rmsd. The structures can be colored to show the values of such numerical attributes, as in the tutorial: Coloring by Sequence Conservation. See also the video Coloring Aligned Proteins by Cα RMSD.

A seq_identity residue attribute is also created automatically (regardless of headers) when a structure is associated with an alignment of at least two sequences. It is the percentage of a column that contains the most common nongap character in that column, and it does not depend on the type of the associated residue(s). See also: sequence refreshAttrs

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Regions (Colored Boxes)

Sequence regions are colored boxes or outlines that enclose one or more residue symbols. A single region may contain any number of disjoint and/or abutting boxes. Pausing the cursor over a region (but not directly over a residue symbol) shows its name in a pop-up balloon.

Some regions are created automatically, such as by association with a structure. Regions can also be created manually by dragging within the sequence window. Manually created regions are shown in this color (default, see Regions settings). The display and colors of existing regions can be controlled with the Region Browser.

The active region is the region most recently clicked, created by dragging, or designated as Active in the Region Browser. The active region is indicated with a dashed outline, and the corresponding parts of any associated structures are selected. Shift-dragging with the left mouse button adds to the active region, whereas Ctrl-dragging creates a new region and makes it the active region. (On Mac, command key replaces Ctrl.) Backspace/delete deletes the active region. Clicking the active region deactivates it, and clicking a different region deactivates the former active region and makes the new region active. A region with no interior color is only responsive to clicks on its borders. Where regions overlap, only the highest is responsive to clicks.

← Region Browser

The Region Browser can be opened by choosing Annotations... Regions... from the context menu.

Regions applicable to the entire alignment or to any of the individual sequences in the alignment can be listed using the pulldown menu near the top of the dialog. Regions applicable to the entire alignment include the ChimeraX selection and any hand-drawn regions, even if they only enclose parts of a single sequence. Regions applicable to an individual sequence include UniProt sequence features and regions created by structure association.

Region Browser columns:

• Active – whether the region is the active region
• Shown – whether the region is shown in the sequence window (however, it could still be obscured by overlapping regions)
• Border – whether the region border color is shown
• region border color (a color well; click to change the color interactively)
• Interio – whether the region interior color is shown
• region interior color (a color well; click to change the color interactively)
• Name – region name
• RMSD (entire-alignment regions only) – region overall RMSD, if applicable, calculated using one point per residue. An overall RMSD is calculated for each region in which a block encloses at least one column associated with at least two structure residues. All pairwise within-column distances are included in the calculation of the overall RMSD, and all distances are weighted equally (columns may not be weighted equally, as they may be associated with differing numbers of structure residues).
• Start (individual-sequence regions only) – position in alignment numbering of the first residue in the region
• End (individual-sequence regions only) – position in alignment numbering of the last residue in the region
• Details (individual-sequence regions only) – additional information about the region from UniProt sequence features

If Include gaps is turned off (unchecked), the chosen region(s) will be drawn to enclose only residues, not gap positions. Although a given region may then appear as disjoint blocks, it will still be a single region.

Since regions may overlap, a region can be considered higher or lower than another. When the mouse focus is in the sequence window, the up arrow and down arrow keys can be used to raise and lower the active region, respectively, and pressing the Delete key will delete the active region. Alternatively, Region Browser buttons can be used to act on the chosen region(s):

• Raise – place region on top (in front) of any overlapping regions in the sequence window and move it to the top of the list in the Region Browser
• Lower – place region below (behind) any overlapping regions regions in the sequence window and move it to the bottom of the list in the Region Browser
• Duplicate – create a copy of the region with a new name
• Rename – change region name
• Delete – remove region; the ChimeraX selection region cannot be deleted, but can contain zero residues
• Info – report region contents in the Log, in terms of alignment numbering, individual-sequence numbering, and residue numbers within any associated structures

← UniProt Sequence Features

Sequences fetched from UniProt may include several annotations. These “sequence features” are automatically assigned as regions in the sequence window. The features are hidden by default, but a dialog for browsing and displaying them appears when the sequence is first opened. This dialog can be reshown at any time by choosing Annotations... Sequence Features... from the context menu.

Choosing a feature type on the left side of the dialog shows the regions for all features of that type; if multiple features of that type are listed on the right, clicking (choosing) any one shows just the region for the individual feature. When the dialog has mouse focus, starting to type the name of a feature type instantly chooses that entry. The up and down arrow keys can also be used to navigate the dialog. Feature listings may include links to evidence code descriptions in the Evidence & Conclusion Ontology and/or sequence variant entries in dbSNP.

The border and interior colors of the currently shown feature region(s) can be changed by clicking the color wells and using the system color editor, and checkboxes allow leaving either the border or the interior uncolored. Region colors can also be changed using the Region Browser. The initially assigned colors may vary between different instances of opening the same data.

If a structure is associated with the sequence, the corresponding structure residues are listed in the lower right area of the dialog. For example, the figure shows all metal ion-binding site regions in UniProt sequence cah1_human, corresponding to residues 64,67,94,96,119, and 200 in chain A of the associated structure, PDB 1bzm. A checkbox option allows automatically selecting the associated structure residues (if any) as features are chosen.

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Sequence-Structure Association

A sequence that arose from a structure's chain information is automatically associated with that chain, with regions for protein structure helices and structure strands. Such sequences are closed when their structure chains are deleted or closed, and they are not automatically associated with other structure chains.

When sequences and structures are opened independently, a structure chain will be associated automatically with a sequence if their sequences can be aligned without mismatches exceeding 1/10 of the residues in the structure chain. The association and number of mismatches will be reported in the Log. For automatic association,

• Gaps in the structure sequence (relative to the sequence opened independently) are not allowed except where residues are missing from the structure, such as in disordered regions with poorly defined density.
• Insertions in the structure sequence relative to the sequence opened independently are not allowed.

For manual association, both of the above are allowed, but only the “extra” residues in the structure not associated with any positions in the sequence (minus the length difference if the structure sequence is longer) are counted along with mismatches and reported in the Log.

Associations can be controlled manually (forced or removed):

• with the sequence command
• by choosing Structure... Associations... from the context menu. The resulting dialog allows associating specified structure chains with specified sequences, or their best-matching sequences determined automatically, or no sequences (dissociation). One or more structure chains can be chosen (highlighted) in the list by clicking and dragging with the left mouse button; Ctrl-click (or command-click if using a Mac) toggles whether a row is chosen.

In the sequence window, regions may be created to show mismatches and missing residues according to the association. If a mismatch or missing residue only occurs in a subset of multiple associated chains, a pink box or gray outline is shown instead (default, see Regions settings).

Further effects of association:

• a color swatch matching the associated model is shown around the sequence name, or a dark-green dashed outline if chains from multiple models are associated
• pausing the cursor over the sequence name reports the association, and over residues in the sequence window reports the corresponding structure residue(s)
• clicking or dragging to update the active region will select the corresponding structure residues
• selections (by any means) of the structure are shown as the region named ChimeraX selection (default, see Regions settings)
• various attributes may be assigned to the structure residues as described under Headers and Residue Attributes

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Sequence Viewer Settings (Preferences)

Choosing Settings... from the Sequence Viewer context menu shows its settings in a separate window, with sections:

• Appearance
• Headers
• Regions

The settings window can be manipulated like other panels in the ChimeraX interface (more...).

Save saves the current settings as preferences, Reset replaces the current settings with the initial “factory” defaults (values shown in bold below), and Restore restores values that were saved previously. The Buttons below... option indicates whether these buttons should apply only to the currently shown section (e.g., Appearance) or to all of the Sequence Viewer preferences. Although there can be multiple settings windows with different values for multiple Sequence Viewer windows, there can be only one set of saved preference values.

← Appearance – how text is displayed in the Sequence Viewer window

• Single Sequence
  • Horizontal spacing (pixels) [S] – space between characters within a row (initial default −2)
  • Wrap – whether to wrap lines within the window (on)
  • Wrap lines at multiple of [N] – when wrapping, allow breaks at multiples of N residues (5)
  • Vertically separate wrapped lines (off)
• Alignment
  • Horizontal spacing (pixels) [S] – space between characters within a row (initial default 0)
  • Wrap if [M] sequences or fewer – do not wrap if the alignment contains >M sequences (8); set to ≤1 for no wrapping
  • Wrap lines at multiple of [N] – when wrapping, allow breaks at multiples of N alignment positions (5)
  • Vertically separate wrapped blocks (on)
  • Show numbering at startup – whether to show alignment numbering (1, 11, etc.) for subsequently opened alignments (on)

← Headers – rows of information above the sequences in an alignment (more...)

• Consensus
  • Show initially (initial default off)
  • Hide low-conservation letters (initial default off) – whether to leave the consensus sequence blank at positions where no type meets the High-conservation threshold; otherwise, the most prevalent type at each position will be shown, even if it appears in fewer than half of the sequences, and if several residue types are equally the most common, one will be chosen at random
  • High-conservation threshold (initial default 0.8) – fraction conservation for showing the consensus type as a purple or red capital letter, with red indicating that all sequences have the same type (a fraction of 1.0); consensus types with a fraction below the threshold will be shown as lowercase letters or (if Hide low-conservation letters is on) as blanks
  • Ignore gap characters (initial default off) – whether to exclude gaps from being considered the most prevalent type at a position; regardless of this option, however, gap positions are always included in the denominator for calculating the conservation fraction

• Conservation
  • Show initially (initial default on)
  • Style
    • identity histogram – what proportion of the sequences have the most prevalent nongap character per alignment column. Histogram bar heights are proportional to (M-1)/(N-1), where M is the number of occurrences of the most prevalent residue type at a position in the alignment and N is the number of sequences in the alignment. Thus, if every sequence has a different residue at a given position, the bar height is zero, not 1/N. However, M/N values (not the values used for display purposes) are used for the seq_conservation attribute.
    • Clustal characters – as in Clustal format,
      • * (asterisk) for complete identity
      • : (colon) for strong group conservation
      • . (period) for weak group conservation
      • blank otherwise
    • AL2CO (ALignment 2 COnservation, initial default) – more sophisticated options for calculating conservation. Users should cite:

      AL2CO: calculation of positional conservation in a protein sequence alignment. Pei J, Grishin NV. Bioinformatics. 2001 Aug;17(8):700-12.

      Conservation values from AL2CO are in standard deviations from the mean (Z-scores) and can range from –∞ (least conserved) to +∞ (most conserved). For purposes of histogram display only, the values are mapped to bar heights 0-1, with conservation values of zero giving a histogram bar height of 0.5.

      AL2CO parameters:

      • Averaging window (initial default 1) – window width, or how many alignment positions to average in a sliding window. The result is assigned to the central column of the window when the width is odd, the column left of center when the width is even (e.g., the fourth position in a window of 8), with various adjustments at the termini. The authors of AL2CO recommend a width of 3 for motif analyses.
      • Conservation measure – which AL2CO conservation equation to use:
        • entropy-based (initial default)
        • variance-based
        • sum of pairs
      • Frequency estimation method – whether/how to weight the sequences
        • unweighted
        • modified Henikoff & Henikoff
        • independent counts (initial default)
      • Gap fraction (initial default 0.5) – maximum fraction of sequences with gaps in a column for conservation to still be calculated; for example, if the gap fraction is 0.25, conservation values will only be calculated for columns in which at least three-fourths of the sequences have residues
      
      Additional settings if the AL2CO Conservation measure is sum of pairs:
      • Matrix [choices: BLOSUM-30, BLOSUM-35, BLOSUM-40, BLOSUM-45, BLOSUM-50, BLOSUM-55, BLOSUM-60, BLOSUM-62 (initial default), BLOSUM-65, BLOSUM-70, BLOSUM-75, BLOSUM-80, BLOSUM-85, BLOSUM-90, BLOSUM-100, BLOSUM-N, Grantham, HSDM, identity, Nucleic, PAM-40, PAM-120, PAM-150, PAM-250, SDM] – which residue similarity matrix to use
      • Matrix transformation – whether/how to modify the matrix
        • none (initial default)
        • normalization     S'_ab = S_ab / (S_aaS_bb)^½
        • adjustment     S''_ab = 2S_ab – ½(S_aa + S_bb)
  
  
• RMSD – per-column root-mean-square displacement among associated structure residues
  • Show initially (initial default off)
  • Atoms used for RMSD
    • Cα/C4' (initial default) – Cα for peptides, C4' for nucleic acids
    • backbone – peptide N, CA, C, O or nucleic acid phosphoribosyl backbone, not including any hydrogens

  When more than one chain per model is associated with the same alignment, the RMSD calculation uses only the chain that gives the lowest overall RMSD.

← Regions – creation and display of regions (colored boxes)

The Regions section of settings controls whether different types of regions should be shown, and if so, their initial colors. Each color well can be clicked to choose a color interactively. On/off checkboxes indicate whether to color the region border (box outline) and/or interior (box fill), whereas full and partial refer to whether the region applies to all structures associated with a position or to only some fraction of those structures.

• New-region border (initial default off, no outline)
• New-region interior (on)
• Show selection region (on)
• Selected-structure border (off, no outline)
• Selected-structure interior (on)
• Show missing-structure regions (on)
• Missing-structure border  full  partial (both on)
• Missing-structure interior  full  partial (both off)
• Show structure-mismatch regions (on)
• Structure-mismatch border  full  partial (both off)
• Structure-mismatch interior  full  partial (both on)