Computational protocol: Hinge Atlas: relating protein sequence to sites of structural flexibility

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Protocol publication

[…] Prior to generating the manually annotated Hinge Atlas, we used computational methods to generate a dataset of hinge residues for our statistical studies. We began by running FlexProt[], a leading hinge identification tool, on all morphs (pairs of homologous protein structures) in the Database of Macromolecular Motions[,,,,,,] FlexProt works by matching and structurally aligning fragments in one structure with corresponding fragments in the other. The goal is to find fragment pairs which (1) have minimal RMSD and (2) are maximal in size. The hinges are then reported as the boundaries separating those fragments. Goal (2) is equivalent to minimizing the number of these hinges. Since domains are never completely rigid, RMSD tends to grow with fragment size and therefore goal (1) is in conflict with goal (2). This conflict is dealt with by providing the user with a series of adjustable parameters, and further by reporting not one but several alternative hinge locations from which the user can choose. We used a combination of computer and manual culling to select those morphs for which the identified hinges met the following criteria:1. Motion was domain wise, i.e. two or more domains could be observed moving approximately as rigid bodies with respect to each other.2. The identified hinge was located in the flexible region connecting two rigid domains, rather than in the domains themselves.3. The morph trajectory was sterically reasonable, i.e. chains were not broken in the attempt to interpolate motion.We found that FlexProt's Maximal[,] RMSD (Root Mean Square Deviation) parameter had a strong effect on the results. Therefore when FlexProt gave visibly incorrect results for a given morph, we reran the program, systematically varying this parameter. If one of these runs gave sufficiently accurate results, the annotation for that morph was entered into the database. We discarded immediately those morphs that did not exhibit clear hinge bending motion. Lastly, we removed redundant morphs using nrdb90[].Note that the definition of a hinge given in the introduction allows for a hinge of zero length. FlexProt indeed often returned such hinges. To deal with this, in all cases one residue on each side of the hinge, was taken to also belong to the hinge. Thus most hinges are two residues long. At the end of this process, the computer annotated set contained 273 morphs.As described, the computer annotation of hinges requires significant human intervention and the results were often debatable. Many of the hinge annotations differed slightly but visibly from the boundary between rigid domains, such that the backbone flexions that could account for the domain motion were not seen in the predicted hinge region. In other cases hinges were missed, and some annotations appeared where no hinge existed. The more flagrantly misannotated hinges were removed from the dataset, but making the manual culling too stringent would simply have resulted in a dataset too small to be statistically meaningful. For these reasons, the computer annotated dataset was not used in most of this work. Nonetheless, the computer annotated dataset is arguably more objective then the manually annotated set described below, and so is made available to the community.To address the accuracy issues, we decided to generate a manually annotated set of hinges – the Hinge Atlas. To generate this set we first created the Hinge Annotation Tool which can also be used by the public as we will now explain. [...] The creation of publicly accessible tools for manual annotation of hinges involved significant changes to the morph page. The morph page is the primary point on MolMovDB[] for analyzing single morphs. It is accessible from the "movies" page or through our search tool, both linked to or visible on our front page. Our server also provides a link to this page in an email sent to the submitter of each morph request. We added all of the new tools to the "Hinge Analysis" tab on this page. The first of these is the Hinge Annotation tool. Each of three rows of "arrow" buttons on this tool move a highlighted window of two residues along the protein chain, allowing the user to highlight up to three hinges in a protein. The "Show all" button then highlights all selected residues in the Jmol viewer window. Once the user is satisfied with the hinge selection, clicking "Submit" records this selection in the database. Once the morph page is regenerated, a "Show public hinge" button will be visible which, when clicked, highlights the selected residues. Lastly, the user can use a pointing device to reorient the protein in the Jmol window to his/her liking. A GIF image based on that view can be generated by clicking on the "color by domain" link. The animation will be rendered using VMD's[] "new cartoon" style, with the identified hinge region and two rigid domains each colored distinctively. The hinge annotations made in this way persist in our database for visualization and use by others, until overwritten. With minor modification, these tools were used to generate the Hinge Atlas dataset of manually annotated hinges. The criteria we used for selection are described in the following section.Highlighting the Hinge Atlas hinges (described below) on the animated morph movie is a matter of going to the morph page and clicking on the "Hinge Analysis" tab as above and clicking the "Show Hinge Atlas hinge" button. The annotated hinge location will be rendered in green spacefill style, which contrasts with the white trace used elsewhere in the protein. […]

Pipeline specifications

Software tools FlexProt, VMD
Application Protein structure analysis