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Cn3D 4.0: Coupling Alignments to Structure
NCBI has released version 4.0 of Cn3D, a program for viewing and manipulating the 3D structures of macromolecules. Cn3D 4.0 is a thorough redesign of the previous version with a growing focus on using structural information as a guide to creating protein multiple sequence alignments. Many new features are summarized below as they apply to the enhanced display of structural data and the new tools for editing and creating protein multiple sequence alignments.
| Click on figure to view enlarged version |
Figure 1: Cn3D 4.0 display shows a structural alignment of two human tyrosine kinases, 1BYG and 1FG1, as computed by the VAST algorithm. The structures include a bound inhibitor, shown in a spacefilling representation along with all atoms within a radius of 5 angstroms in a ball and stick representation. The alignment viewer displays a portion of the structural alignment, with aligned residues in capital letters and each aligned block represented in the bar above the sequences.
Editing Multiple Sequence Alignments
While previous versions of Cn3D allowed users to import sequence alignments into the program, Cn3D 4.0 allows users to edit these alignments. Alignments in Cn3D consist of multiple blocks of ungapped sequence, each ideally representing an element of the 3D structure such as an individual alpha-helix or beta-strand. In the new Editor mode of the Alignment Viewer, the pre-existing blocks of an imported alignment can be expanded, contracted, or deleted, and new blocks can be created using a “block editor”, visible whenever editing is enabled. A given block can also be split into two blocks, or two blocks can be merged into one. One can set the coloring options so that these changes to the alignment are immediately reflected in the color of the affected residues on the displayed structure. One can also manually change the alignment of any sequence within a block by dragging the residues left or right. To assist with these tasks, the Alignment Viewer can search for a sequence pattern using Prosite syntax. Figure 1, on page 1, displays a VAST (Vector Alignment Search Tool) structural alignment of four tyrosine kinases, and the Alignment Viewer clearly indicates the block structure of the alignment.
Creating New Sequence Alignments
In addition to editing existing sequence alignments, users can now create new alignments and add new sequences to existing alignments. Both of these functions are provided in a Cn3D window named the Import Viewer. The Import Viewer serves as a workspace in which users import sequences, either over a network or from a local disk, pairwise align them to the master sequence of the currently loaded alignment, and then merge the new alignments into the Alignment Viewer. Sequences from the Alignment Viewer can also be sent to the Import Viewer for realignment if desired. Users can choose between three algorithms with which to align their imported sequences: BLAST, PSI-BLAST, or sequence-structure threading, the latter two of which are new to Cn3D 4.0. In PSI-BLAST, Cn3D 4.0 will calculate a Position Specific Scoring Matrix (PSSM) from the currently loaded alignment, and use this matrix to generate an alignment of the imported sequence to the master. Threading employs an algorithm developed at NCBI to align the imported sequence to the master using both structure and sequence information generated from a user-controlled combination of the PSSM and residue contact potentials. This combination of algorithms gives the user a powerful set of tools to correlate sequence and structure conservation.
Enhanced Molecular Display
Upon opening a structure record in Cn3D 4.0, users will notice improvements in both the quality and speed of the graphical display. In addition, the manipulation of the structure is now more straightforward and responsive. For example, zooming the view in or out can now be performed by dragging the mouse while holding down a control key (Ctrl on PCs, Cmd on Macs), and translation of the structure is likewise performed while holding down the Shift key. Moreover, Cn3D makes animating molecules easier by allowing the user to step through multiple structures in the display with simple keystrokes. The latter is especially useful for NMR ensembles, structural alignments, or crystal structures with multiple sets of coordinates.
Cn3D’s menus have been rede-signed to provide easier access to the numerous display options. The Color and Style menus are combined into a new Style menu that collects commonly used options and provides direct access to dialog boxes controlling rendering details. In keeping with the major updates in handling alignments, a new Show/Hide menu gives convenient access to commands controlling the display of aligned versus unaligned portions of the displayed structure.
Cn3D 4.0 now features a Select by Distance option that allows users to find all atoms within a chosen radius, in Angstroms, from currently selected atoms. The residues found are highlighted both in the structure and sequence views for ease of identification. This option is especially useful for exploring contacts between a ligand and binding site residues, or between adjacent units of secondary structure. In Figure 1, the residues within 5 Angstroms of the two tyrosine kinase inhibitors are shown in a ball and stick representation.
In the near future, users should expect the release of the curated Conserved Domain records that will be viewed using Cn3D. Cn3D will not only show the alignments, but also curated annotations on both the sequences and structures, highlighted substructures and alignment columns of functional importance, and links to PubMed literature. These unique records will provide a wealth of critical biochemical data about a protein family in a single, integrated view.
