This help document describes how to use VAST+ , an enhanced version of VAST that finds macromolecular structures that are similar in 3D shape, and groups them as complete or partial matches to the biological unit (" biounit ") of a query structure, making it possible to find similar macromolecular complexes. Original VAST , which focuses on 3D similarities between individual protein molecules or 3D domains (substructures) rather than biological units, is described in the original VAST Help document . The VAST Search tool , which accepts input of a query structure's 3D coordinates and returns original-style VAST results, is described in the VAST Search Help document . What is VAST+ ? V ector A lignment S earch T ool Plus Comparison of original VAST vs. VAST+ How can VAST+ be used to learn more about proteins? Illustrated examples of VAST+ results for: Glutamate Dehydrogenase ( Thermotoga maritima ) Deoxyhemoglobin ( Homo sapiens ) Precalculated results PDB ID or MMDB ID Enter ID directly into search box on VAST Home page Structure or Protein database search : 3D structure → similar structures protein sequence → structure → similar structures protein sequence → related structures → similar structures input 3D coordinates of query structure into VAST Search page Query structure (summary information) Interactions schematic Molecular graphic MMDB ID , PDB ID Biological Unit Source Organism Number of proteins Number of nucleotides Number of chemicals Similar structures Folder tabs to select 3D alignment type: All matching molecules superposed Invariant substructure superposed Filters to subset the search results by: Number of aligned protein molecules complete match to biological unit of query structure partial match to biological unit of query structure Filter results by superkingdom Ranked list of hits Aligned proteins Aligned residues Sequence identity View details for a similar structure Search within results "Original VAST" button View original style VAST results (by individual protein molecule) "No matched structure found" (null result) "New Search" button Enter a new PDB ID or MMDB ID to find similar structures Detailed Views/Graphical Displays for Individual Hits Detailed view comparing the query structure and a matched structure Aligned molecules Interaction schematic of query structure vs. matched structure List of matching protein molecules Sequence Alignment Views Align one pair of matching molecules Display all aligned molecule sequences Superposition of query and neighbor in 3D Cn3D version for viewing VAST+ superpositions (important note) Original style VAST display (example) similarities between individual protein molecules or 3D domains VAST+ Algorithm Overview of method used to identify similarly shaped macromolecular complexes Illustration : VAST+ algorithm Additional Details 3D alignment of biological units (macromolelcular complexes) initial alignment (all matching molecules superposed) refined alignment (invariant substructure superposed) minimum size of structures that are neighbored maximum size of structures that are neighbored Log of Changes to VAST+ Citing VAST and VAST+ Additional References VAST+ (Vector Alignment Search Tool Plus ) Definition Comparison of original VAST vs. VAST+ Focus on 3-dimensional similarities between individual protein molecules , or between individual 3D domains . (Original VAST lists each protein molecule and 3D domain in the asymmetric unit of the query structure, and links to structures that are similar in shape to the protein molecule or 3D domain you select.) illustrated example... Focus on 3-dimensional similarities between macromolecular complexes ( biological units ). illustrated examples... View the superposition of two *or more* protein molecules at the same time. View the superposition of two macromolecular complexes (The latest version of NCBI's structure viewing program, Cn3D 4.3.1, is needed in order to do this.) Align (in 3D space) two or more proteins from the same structure record . Rank structures by their degree of similarity to the query protein's biological unit . Align (in 3D space) two or more domains from the same molecule to each other, for example, to compare repeats. (For example, compare the first and second immunoglobulin (Ig) domain in a protein molecule.) Compare different states of the same oligomer . (For example, compare the conformation of the deoxygenated and oxygenated states of hemoglobin.) How can VAST+ be used to learn more about proteins? Find protein complexes that have the same oligomeric state and similarly shaped proteins , regardless of the degree of sequence similarity. Example: view the VAST+ neighbors for 4MGG : Crystal Structure of an Enolase (Mandelate Racemase Subgroup) From Labrenzia Aggregata IAM 12614 (Target Nysgrc-012903) With Bound MG, Space Group P212121. (Included among the VAST+ results are structures such as 3SN4 , which has remarkable conservation of the overall complex structure, despite very low sequence similarity.) Example: view the VAST+ neighbors for 1B26 : Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium thermotoga maritima at 3.0 Å resolution. (Included among the VAST+ results are structures such as 1GTM , which has a similarly shaped proteins and a hexameric biological unit , but a somewhat different configuation of the hexamer when compared with the query structure, as shown in the illustrated example below.) Compare the conformation of different states of the same oligomer , such as oxy- and deoxyhemoglobin. Example: view the VAST+ neighbors for 1HBB : High-resolution X-ray Study of Deoxyhemoglobin Rothschild 37beta Trp-> Arg: a Mutation That Creates an Intersubunit Chloride-binding Site. (Included among the VAST+ results are structures such as 1GZX , an oxy T State hemoglobin, which can be viewed as a 3D superposition on the query structure, to compare configurations, using Cn3D 4.3.1 .) Compare the binding interfaces of a query structure and its nearest "neighbors," such as the differences in the binding interface among similarly shaped immunocomplexes . Example: view the VAST+ neighbors for 3O6F : Crystal Structure of a Human Autoimmune TCR Ms2-3c8 Bound to MHC Class II Self-ligand Mbphla-dr4. (Included among the VAST+ results are structures such as 1J8H , which contains a complex between HLA-DR3, an Influenza hemagglutinin peptide, and a human alpha/beta T-cell receptor. This demonstrates how well the auto-reactive T-cell receptor complex mimics complexes that include foreign peptides.) Illustrated examples of VAST+ results: Illustrated Example: VAST+ results for Glutamate Dehydrogenase ( Thermotoga maritima ) VAST+ search results for 1B26 Glutamate Dehydrogenase ( Thermotoga maritima ) , as of 02 October 2014, with detailed view of match to 1GTM . Click anywhere on the image to open a live web page with current VAST+ results for 1B26 . Illustrated Example: VAST+ results for Deoxyhemoglobin ( Homo sapiens ) VAST+ search results for 1HBB High-resolution X-ray Study of Deoxyhemoglobin Rothschild 37beta Trp-> Arg: a Mutation That Creates an Intersubunit Chloride-binding Site , as of 02 October 2014, with detailed view of match to 1FSL . Click anywhere on the image to open a live web page with current VAST+ results for 1HBB . VAST+ results are precalculated for structures in MMDB PDB ID or MMDB ID Enter the PDB ID or MMDB ID for the structure of interest directly into the VAST+ home page . VAST+ will then display your query structure followed by a list of geometrically similar structures, ranking them by the extent of their similarity to the query structure's biological unit . A detailed view for each retrieved structure shows the correspondence between individual protein molecules in the query and subject structures. The " 3D View " button enables you to view a superposition of the aligned regions. Please note that the latest release of Cn3D ( version 4.3.1 ) is necessary in order to properly view the superpositions. Structure or Protein database search 3D structure → similar structures Search the Entrez Structure database for a text word or phrase of interest. For example, retrieve structures that contain the term immunoglobulin , or the phrase "tumor suppressor" , anywhwere in the record. If desired, you can limit your query to a specific search field of the record, such as the title of the article describing the structure, for example: "tumor suppressor"[title] or immunoglobulin[title] . (The MMDB Help document provides search tips for the structure database.) On the Structure search results page , click on the thumbnail image or title of a structure of interest to open its summary page , then press the " Similar Structures: VAST+ " button near the upper right hand corner of the page. The resulting page will display the VAST+ search results , listing structures that are geometrically similar to the structure you checked or viewed, ranked by the extent of their similarity to the query structure's biological unit . protein sequence → structure → similar structures Search the Entrez Protein database for a text word or phrase of interest. (For tips on searching the Entrez Protein database, see Entrez Help , Entrez Sequences Help , and Entrez Nucleotide and Entrez Protein FAQs .) On the Protein search results page , click on the title of a protein of interest to open its sequence record, then scroll down to the " Related Information " section in the right hand margin of the page. If the protein sequence was derived from a 3D structure record , the Related Information section will include " Structure ." Select that option. The resulting page will display the 3D structure record from which the protein sequence was derived . From there, follow the link for " Similar Structures: VAST+ " to see a list of geometrically similar structures identified by VAST+ . Note: If the "Related Information" section of a protein sequence record does not list "Structure" as an option, then use the next method described below, to traverse from protein sequence → related structure → similar structures . protein sequence → related structures → similar structures Only a small percentage of sequence records in the Entrez Protein database are derived from 3D structure records. However, it is still possible to find 3D structure records for the majority of protein sequence records by retrieving structures that are sequence-similar to your protein of interest. These are called " Related Structures " and they have been identified by a program called CBLAST . From there, you can find geometrically similar structures that have been identified by VAST. To do this, follow these steps: Search the Entrez Protein database for a text word or phrase of interest. (For tips on searching the Entrez Protein database, see Entrez Help , Entrez Sequences Help , and Entrez Nucleotide and Entrez Protein FAQs .) On the Protein search results page , click on the title of a protein of interest to open its sequence record, then scroll down to the " Related Information " section in the right hand margin of the page. An option for " Related Structures " will be present if there are 3D structures that are sequence-similar to the protein currently displayed. Select that option. The resulting page will display a list of 3D structures that are sequence-similar to your protein of interest. Click on the thumbnail of any structure to open its summary page . From there, follow the link for " Similar Structures: VAST+ " to see a list of geometrically similar structures identified by VAST+ . Note: If the "Related Information" section of a protein sequence record also lists "Structure" as an option, that means the protein sequence was derived from a 3D structure record. In that case, you can also use the previous method for accessing 3D structures, if desired, to traverse from protein sequence → structure → similar structures. 3D coordinates of a query structure The VAST+ search results page displays summary information about your query structure , followed by a list of similar structures that are ranked based on their degree of similarity to the query structure's macromolecular complex ( biological unit ) ( illustrated examples ) . By default, all similar structures are listed, starting with complete matches to the query structure's biological unit, followed by partial matches , and ending with matches to individual protein molecules. If desired, you can activate a subset of filters in order to display only the subset of similar structures that meet the specified criteria. It is also possible to change the sort order of the similar structures, and to search within results to see if/where a specific structure falls within the search results. Click on the plus button beside any structure to see a detailed view comparing the query structure to a matched structure . (If you prefer to view your search results in the original style VAST display, which lists structures that have similarities to individual protein molecules, or similarities to individual 3D domains , in your query structure, click the " Original VAST " button.) A VAST+ search results page includes the following components: Query structure (summary information) Similar Structures Folder tabs to select 3D alignment type:

All matching molecules superposed Invariant substructure superposed Filters to subset the results by: number of matching molecules complete match to biological unit of query structure partial match to biological unit of query structure Ranked list of hits : a table listing similar structures ranked by the extent of their similarity to the query structure's biological unit. Aligned residues Sequence identity View details for a similar structure Search within results "Original VAST" button "No matched structure found" "New Search" button All matching molecules superposed (initial alignment) When you click on the plus button beside any structure to see a detailed view/graphical display comparing the query structure to the matched structure, the " visualize 3D structure superposition " button will open the initial 3D alignment , with all matching molecules superposed . The VAST algorithm section of this document provides more details about the initial alignment as well as an example. Invariant substructure superposed (refined alignment) When you click on the plus button beside any structure to see a detailed view/graphical display comparing the query structure to the matched structure, the " visualize 3D structure superposition " button will open the refined 3D alignment , with the invariant substructure superposed . The VAST algorithm section of this document provides more details about the refined alignment as well as an example. Filter by number of matching molecules Complete match to biological unit of query structure -- Each of the biopolymer molecules in the biological unit of the query structure have a match (i.e., are similar in 3D shape) to a biopolymer molecule in the subject. The illustrated examples of VAST+ results include complete matches, which are indicated by solid red circles . Partial match to biological unit of query structure -- A subset of the biopolymer molecules, or an individual biopolymer molecule, from the biological unit of the query structure have a match (i.e., are similar in 3D shape) to a biopolymer molecule in the subject. The illustrated examples of VAST+ results include partial matches, which are indicated by half-filled red circles . Additional notes about matching molecules: Biopolymers refer to protein , DNA , and RNA molecules. At this time, VAST (and VAST+) operates only on protein molecules . Some biological units might contain other types of biopolymers as well, such as DNA or RNA molecules. VAST and VAST+ do not currently compute similarities between the 3D shapes of DNA molecules, or similarities between the 3D shapes of RNA molecules. Example : 1TUP , "Tumor Suppressor P53 Complexed With DNA," has a pentameric biological unit composed of three proteins and two nucleotide sequences. VAST will find other structures that have similarly shaped proteins, but will not take the DNA molecules into consideration. Therefore, there will be no "complete match" structures in the VAST+ search results for 1TUP , as it is not currently possible to find a match for all five molecules in the biological unit. The top scoring hits, which match all three protein molecules in 1TUP, are nevertheless easy to find as they are displayed at the top of the "partial match" results, based on the default sort order . Mimimum size of protein: VAST does not act upon protein molecules that have fewer than three secondary structure elements (SSE's: alpha helices and/or beta sheets) because they can generate spurious hits. Such short proteins are therefore not considered in VAST+ calculations or results. For example, if a pentameric structure contains four large proteins, plus one small protein that doesn't meet the VAST size requirements, VAST will only operate on the four large proteins from the biological unit. Therefore, there will be no "complete match" structures in the VAST+ search results, and the top hits will be, at the most, structures that match all four long proteins. Example : 2YPL , "Structural Features Underlying T-cell Receptor Sensitivity to Concealed MHC Class I Micropolymorphisms," contains four human proteins, plus one very small HIV protein. None of the structures in the VAST+ search results for 2YPL are listed as a complete hit, because the fifth protein in the 2YPL's biological unit does not meet the minimum size requirements for VAST. So the best possible match that VAST (and VAST+) can find is to 4 out of 5 proteins in the query structure's biological unit, and the top hits will be structures that, at the most, match all four of the long proteins. Each of those hits will be listed as a "partial match." Pairwise alignments between individual protein components of the query structure and subject are compared to each other for compatibility, and compatible/matching alignments are clustered into sets of alignments that together constitute a biological unit match. Pairwise alignments are compatible: (1) if they do not share the same macromolecules, i.e., an individual protein molecule from the query structure structure cannot be aligned to two or more protein molecules from the subject at the same time; and (2) if they generate similar instructions (spatial transformation matrices) for the superpositions of co-ordinate sets. A simple distance metric can be used to compare transformation matrices, and it lends itself to cluster alignment sets efficiently. Filter by Taxonomy Filter results by domain (superkingdom) -- The "Filter by Taxonomy" options enable you to view the subset of similar structures from the "Eukaryota," "Bacteria," or "Archaea" domains (superkingdoms), from "Viruses," or from "Other" groups (such as synthetic constructs). Activate the check box(es) beside the group(s) of interest to see only the desired subset(s) of similar structures. Note that a taxonomy filter will retrieve structures in which all biomolecules in the structure are from the selected superkingdom. For example, the "Viruses" filter will retrieve structures that only contain molecules from viruses, and not molecules from any other superkingdom. Structures that are composed of synthetic constructs, or composed of molecules from two or more superkingdoms, are included in the " Others " taxonomy category. For example, PDB ID 4N9F would be classified with a taxonomy of "Others" because it includes molecules from Human immunodeficiency Virus 1 and Homo sapiens, which fall into the superkingdoms of Viruses and Eukaryota, respectively. Sort Options (Scores) Aligned proteins -- The number of protein molecules in the query structure have a match (i.e., similar 3D shape) to a protein molecule in the subject. If all of the molecules in the query structure match molecules in the subject, then a solid red circle icon indicates there is a complete match to biological unit of query structure. ( illustrated examples of VAST+ results ) If a subset of the molecules, or an individual molecule, from the query structure have matching molecules in the subject, then a half-filled red circle icon indicates there is a partial match to biological unit of query structure. ( illustrated examples of VAST+ results ) RMSD -- The average root mean square deviation (RMSD), in Angstroms (Å), of all aligned residues between the query structure and the subject. This number is calculated after optimal superposition of two structures, as the square root of the mean square distances between equivalent C-alpha atoms. Smaller RMSD values indicate greater structural similarity. (Note that the RMSD value scales with the extent of the structural alignments and that this size must be taken into consideration when using RMSD as a descriptor of overall structural similarity.) The RMSD values displayed in VAST+ results might be much higher than those displayed in Original VAST results because the 3D alignment is optimized for the whole assembly rather than for individual protein molecules. Specifically, VAST+ takes all of the individual 3D domain alignments and calculates a global superposition. To do this, individual protein molecules might be shifted from their ideal pairwise superposition, in order to be able to generate a global superposition of all matched molecules between the query structure and the subject. For example, Original VAST might give RMSD values of about 3-4 Å at the worst, and rarely up to 6 Å for very large structures. In contrast, VAST+ results might generate more hits with RMSD values of 5-6 Å or more, and a number of cases with large values such as 20 Å or more . This can be due to flexibility in the biological unit, which might indicate something interesting. An example is the chaperone protein 3J1B : Cryo-em Structure of 8-fold Symmetric Ratcpn-alpha in APO State. The VAST+ neighbors for 3J1B include 3J03 : Lidless Mm-cpn in the Closed State With Atpalfx, with an RMSD value of 13.41 Å. Aligned residues -- The total number of amino acids, from all protein molecules in the query structure's biological unit, that are spatially aligned to the subject. Sequence identity -- The percent of aligned residues that are identical, averaged over all of the aligned protein molecules (rounded to the nearest whole number on the VAST+ results display). Taxonomy -- Click on the up/down arrows in the taxonomy header to sort hits alphabetically by genus name. If you choose to sort by taxonomy, the similar structures are sorted based on the following criteria: (1) alphabetically by genus, then within a genus, the structures are sorted based on (2) number of aligned proteins , (3) RMSD , (4) number of aligned residues , and (5) sequence identity , in that order. (Note: If you would like to only see the subset of search results from a specific superkingdom, use the taxonomy filter to select the desired superkingdom, then click on the arrows in the "Taxonomy" column header of the search results table to sort the structures from the superkingdom alphabetically by genus.) View details for a similar structure Click on the plus button beside any structure to see a detailed view comparing the query structure to the matched structure. A separate section of this help document provides a description of the detailed view . Search within results The " Search within results " text box enables you to enter a PDB ID, MMDB ID, or text word from the title of a structure of interest to find out if it among the similar structures retrieved by VAST, and if so, to instantly take you to the page of results on which it appears, with your structure of interest highlighted {in yellow}. If you are viewing only a subset of the search results (for example, because you have checked a box beside a bar graph of interest to filter the results), and your structure of interest is not in that subset but is in the overall search results, VAST will display a relevant message that includes a link which opens the complete set of search results, automatically highlighting your structure of interest. If, on the other hand, you enter a PDB ID of a structure that is not listed among the VAST results, you will see an error message indicating that your structure of interest was not among the results. Example: Retrieve the VAST results for 1HBB : "High-resolution X-ray Study of Deoxyhemoglobin Rothschild 37beta Trp-> Arg: a Mutation That Creates an Intersubunit Chloride-binding Site (human hemoglobin)." Let's say that you suspect 1UMO ("The Crystal Structure Of Cytoglobin: The Fourth Globin Type Discovered In Man") will be one of the structures retrieve and you quickly want to find where that structure falls within the ranked list of similar structures. To do that, simply enter the PDB ID 1UMO (or its corresponding MMDB ID 66087) in the "search within results" text box, and you will be taken immediately to the appropriate page of search results, with 1UMO highlighted in yellow . You can then click on the plus button to the left of that structure in order to see more details about the comparison between 1HBB and 1UMO. If you enter the PDB ID or MMDB ID of a structure that is not similar (as determined by VAST+) to the query structure, a pop-up message will appear that says, "XXX is not a simlar structure of 1HBB." "Original VAST" button (on the search results page) "No matched structure found" (null result) "New Search" button (on the search results page) Detailed View Comparing the Query Structure and a Matched Structure Interaction schematic of query structure vs. matched structure The interaction schematics for both the query structure and the matched structure (VAST+ neighbor) are displayed side by side. Protein molecules that have a match are designated by a broken (dashed) outline . Protein molecules that do not have a match are shown with a solid outline . Click on the icon of any protein molecule in either one of the interaction schematics to see the protein's name and to highlight the corresponding molecule in the other structure. When you click on an icon, its broken (dashed) outline will change from red to black , and its protein name will be highlighted in yellow . The matching protein in the other structure will be highlighted in the same way. The molecule that is highlighted in the query structure's interaction schematic and protein list is referred to as the selected protein , and is the focal point of the 3D and sequence alignment displays. For example, if you select a protein from the query structure and then press the button for " Visualize 3D structure superposition (with Cn3D app) " or " View aligned sequences ," the 3D view and sequence alignment windows will be centered on the selected protein. You can then rotate the 3D view and scroll through the sequence alignment window to see other proteins, if desired, but the initial view will be centered on the selected protein. List of matching protein molecules Beside each interaction schematic is a list of the proteins that are spatially aligned to a protein in the matching structure. Click on the name of any protein to highlight that protein in both the list and in the interaction schematic. This action will also automatically highlight the name and icon of the matching protein in the other structure. Regardless of how many proteins are present in the biological unit of the query structure or subject, the list of protein names shows only the subset of proteins that have 3D similarity to each other . (You can still see the names of other proteins in the query or subject, however, by clicking on the icons in the interaction schematics.) Sequence Alignment Views VAST does two things for each pair of similar proteins: it calculates an optimal 3D superposition for the conserved core, and constructs a sequence alignment based on the correlation of the 3D structures . The detailed view of a query vs. subject structure comparison (which opens when you click on the plus button beside any hit) allows you to view the pairwise sequence alignments of matching protein pairs. To do this, click on the " View aligned sequences " button that appears beneath the list of matching protein molecules. A separate window will open, showing the pairwise sequence alignments for all matching protein molecules. If you selected a protein from the query structure's interaction schematic or protein list before pressing the "View aligned sequences" button, the sequence alignment display will automatically scroll to the selected protein. The sequence alignments shown in VAST use the following conventions: UPPER CASE LETTERS represent amino acids that are aligned in 3D space, LOWER CASE LETTERS represent unaligned amino acids , and RED LETTERS represent identical amino acids . Because the protein alignments produced by VAST do not require sequence similarity (but instead simply require a common spatial position of amino acids), these views do not necessarily represent alignments of evolutionarily conserved sequence blocks. However, the 3D alignments often coincide with evolutionarily conserved sequence blocks. Superposition of query and similar structure in 3D The detailed view of a query vs. subject structure comparison (which opens when you click on the plus button beside any hit) provides a " Visualize 3D structure superposition " button at the bottom of the table of aligned molecules. That button opens a menu that allows you to choose which program to use for interactively viewing the superposition: iCn3D (Web) - NCBI's WebGL-based viewer that provides interactive views of macromolecular structures and chemicals without the need to install a separate application. Note: To use iCn3D, you just need to use a web browser that supports WebGL . If your browser doesn't support WebGL, you might need to modify the settings in the browser to enable WebGL, or update your web browser to a newer version that supports WebGL. (See the WebGL site for more information about compatibility with various web browsers.) iCn3D's 3D view window will show the complete biological unit of the query structure and its VAST+ neighbor. Click on iCn3D's "View Sequence" button, if desired, to open a sequence view window that shows all pairs of aligned sequences from the query structure and its VAST+ neighbor. Note that iCn3D can show two different types of 3D superpositions: (1) initial alignment , in which all matching molecules are superposed, and (2) refined alignment , in which an invariant substructure is superposed). Cn3D (App) - a helper application for your web browser, available for Windows and Macintosh, which simultaneously displays structure, sequence, and alignment, and has powerful annotation and alignment editing features Note: In order for this selection to work, you must have Cn3D 4.3.1 installed on your computer. Cn3D's 3D view window will show the complete biological unit of the query structure and its VAST+ neighbor. The 3D view will be centered on the selected protein (i.e., on the protein that was highlighted in the query structure's interaction schematic before you pressed the "Visualize 3D structure superposition" button) Cn3D's "Sequence/Alignment Viewer" window will open automatically, showing the selected protein that you chose in the query structure's interaction schematic (or showing structure's first pair of aligned sequences from the query structure and its VAST+ neighbor, if you didn't click on any protein in the interactions schematic). To view a different pair of aligned sequences, you can either: go back to the VAST+ search results page and click on the protein of interest in the query structure's interaction schematic (and then follow the options again to "Visualize 3D structure superposition > Cn3D (app)" or choose the "Select > Pick Structures" menu option in Cn3D and then use CTRL+Click in the list of proteins to select the new protein you'd like to see in the "Sequence/Alignment Viewer" window. The folder tabs on the VAST+ search results page affect which type of alignment will be shown in the 3D view. For example, when you click on the "Visualize 3D structure superposition" button, you will either see: all matching molecules superposed (initial alignment) OR invariant substructure superposed (refined alignment), depending on which folder tab you have selected. The VAST algorithm section of this document provides more details about the initial alignment and refined alignment , as well as an example of each. iCn3D (Web) - NCBI's WebGL-based viewer that provides interactive views of macromolecular structures and chemicals without the need to install a separate application. Note: To use iCn3D, you just need to use a web browser that supports WebGL . If your browser doesn't support WebGL, you might need to modify the settings in the browser to enable WebGL, or update your web browser to a newer version that supports WebGL. (See the WebGL site for more information about compatibility with various web browsers.) iCn3D's 3D view window will show the complete biological unit of the query structure and its VAST+ neighbor. Click on iCn3D's "View Sequence" button, if desired, to open a sequence view window that shows all pairs of aligned sequences from the query structure and its VAST+ neighbor. Note that iCn3D can show two different types of 3D superpositions: (1) initial alignment , in which all matching molecules are superposed, and (2) refined alignment , in which an invariant substructure is superposed). Cn3D (App) - a helper application for your web browser, available for Windows and Macintosh, which simultaneously displays structure, sequence, and alignment, and has powerful annotation and alignment editing features Note: In order for this selection to work, you must have Cn3D 4.3.1 installed on your computer. Cn3D's 3D view window will show the complete biological unit of the query structure and its VAST+ neighbor. The 3D view will be centered on the selected protein (i.e., on the protein that was highlighted in the query structure's interaction schematic before you pressed the "Visualize 3D structure superposition" button) Cn3D's "Sequence/Alignment Viewer" window will open automatically, showing the selected protein that you chose in the query structure's interaction schematic (or showing structure's first pair of aligned sequences from the query structure and its VAST+ neighbor, if you didn't click on any protein in the interactions schematic). To view a different pair of aligned sequences, you can either: go back to the VAST+ search results page and click on the protein of interest in the query structure's interaction schematic (and then follow the options again to "Visualize 3D structure superposition > Cn3D (app)" or choose the "Select > Pick Structures" menu option in Cn3D and then use CTRL+Click in the list of proteins to select the new protein you'd like to see in the "Sequence/Alignment Viewer" window. The folder tabs on the VAST+ search results page affect which type of alignment will be shown in the 3D view. For example, when you click on the "Visualize 3D structure superposition" button, you will either see: all matching molecules superposed (initial alignment) OR invariant substructure superposed (refined alignment), depending on which folder tab you have selected. The VAST algorithm section of this document provides more details about the initial alignment and refined alignment , as well as an example of each. all matching molecules superposed (initial alignment) OR invariant substructure superposed (refined alignment), Original style VAST display lists each protein molecule and 3D domain in the asymmetric unit of the query structure , and retrieves structures that are similar in shape to any individual protein molecule or 3D domain , and enables you to view their sequence alignments and 3D superpositions. Part 1 of the illustration shows colored bars that represent the compact substructures , or 3D domains , detected by VAST in the query structure's protein molecule(s). These 3D domains serve as the fundamental unit of structure comparison. (The data processing : geometrical features section of the MMDB help document provides more information about how the 3D domains and similar structures are identified.) To view the 3D domains that have been identified in a protein molecule, open the MMDB structure summary page for a structure of interest, sroll down to the table of molecules and interactions , and click on the " show annotation " link for the protein of interest. (For example, open the structure summary page for 1PTH .) Part 2 of the illustration shows the original-style VAST display, which consists of a table summarizing the protein molecules and 3D domain in the query structure, and the number of structures that are geometrically similar to each individual protein molecule or 3D domain in your query structure. For any protein molecule or 3D domain of interest, click on the link in the "# of Related Structures" column to view a list of similar structures. The list includes a graphical display of alignment footprints and provides options to view sequence alignments and superposed structures. Example - Original style VAST display (as of 07 November 2013) for 1PTH , "The Structural Basis of Aspirin Activity Inferred From the Crystal Structure of Inactivated Prostaglandin H2 Synthase" (sheep prostaglandin H2 synthase) Open a live web page with original-style VAST results for 1PTH. Overview of method used to identify similarly shaped macromolecular complexes Illustration: VAST+ algorithm NOTE: You can use iCn3D to open an interactive view of the initial alignment of 1HHO and 4N7N , and an interactive view of the refined alignment of 1HHO and 4N7N , which are the sample structures for human oxy- and deoxyhemoglobin that are featured in the illustration above. Additional details about the method used to identify similarly shaped macromolecular complexes MMDB Data Processing : Identifies geometrical features within a structure: secondary structures Identifies biological units within a structure Original VAST : Compares the shape of individual 3D domains and individual protein molecules throughout the Molecular Modeling Database. (Details about the original VAST algorithm are provided in the references noted at the end of this file and in the original VAST help document .) Compares the shape of oligomers throughout the Molecular Modeling Database, using the method illustrated above. Finds structures that have a complete match to the query structure's biological unit Finds structures that have a partial match to the query structure's biological unit Calculates scores , and use these to rank and sort hits by: number of aligned proteins number of aligned residues percent sequence identity Generates a 3D alignment of biological units (macromolelcular complexes), as shown in the 4th and 5th sections of the illustration above Initial alignment (all matching molecules superposed): The initial superposition of a query structure and its VAST+ neighbor uses the complete set of individually aligned macromolecules and corresponding matching amino acids to calculate a superposition of the complex structures . The spatial distance between the structures is then calculated as the root-mean-square deviation (RMSD) , which is a measure of the average distance between the atoms (equivalent C-alpha atoms) of the superposed proteins. For example , when comparing the structures of human hemoglobin in the oxy and deoxy form (1HHO and 4N7N, respectively), a total of 572 amino acids are aligned, with an RMSD of 1.02 Angstroms , as shown in the 4th section of the illustration above. You can use iCn3D to open an interactive view of the initial alignment of 1HHO and 4N7N , which are the sample structures for human oxy- and deoxyhemoglobin that are featured in the illustration above. Note: After opening the interactive view, notice that the iCn3D URL ends with an " atype " parameter, which enables you to specify the desired alignment type . A value of " atype=0 " loads the initial alignment. The iCn3D help document includes additional details about the URL format that can be used to open structures directly in iCn3D, including a list of optional parameters , such as "atype" and more. Refined alignment (invariant substructure superposed): Using the RMSD from the initial alignment as a threshold , VAST+ identifies a subset set of amino acids that have highly similar 3D positions in the query and subject complex structure , and uses it to create a refined alignment that allows identification and visualization of the most similar portion of the structures (the structurally invariant core of the assembly), as well as the differences between the structures. To do this, it finds all amino acids pairs that have a distance equal to, or less than, the original RMSD , and then regenerates the superposition to create a more finely tuned alignment for that subset of amino acids. As a result, the total number of aligned amino acids and the RMSD generally decrease compared to the initial alignment. For example , the refined alignment of human hemoglobin in the oxy and deoxy form ( 1HHO and 4N7N , respectively), has a total of 221 amino acids aligned (compared to the initial 572), and an RMSD of 0.39 Angstroms (compared to the initial 1.02 Angstroms), as shown in the 5th (last) section of the illustration above. The interpretation of the refined alignment is that the alpha-beta dimers are the most stable part of the tetramer, in the oxy- vs. deoxy- tetramers. That is, the main conformational change is a shift in the relative positions of the alpha-beta dimers upon oxygen binding (or unbinding). This can be readily visualized in iCn3D by opening an interactive view of the refined alignment of 1HHO and 4N7N and then toggling between the two structures by clicking the "Alternate Selection" button . Note: After opening the interactive view, notice that the iCn3D URL ends with an " atype " parameter, which enables you to specify the desired alignment type . A value of " atype=1 " loads the refined alignment. The iCn3D help document includes additional details about the URL format that can be used to open structures directly in iCn3D, including a list of optional parameters , such as "atype" and more. In general, there is not going to be a unique subset of amino acids that have the best alignment. For example, in the hemoglobin tetramer case above, one subset (solution to the problem) is going to be (roughly) one of the alpha-beta dimers, another possibility is the other alpha-beta dimer. In either case, the refined alignment allows identification and visualization of the most similar portion of the structures (the structurally invariant core of the assembly), as well as the differences between the structures . Minimum size of structures that are neighbored: VAST, and therefore VASTS+, only operates on protein molecules that contain three or more secondary structure elements (SSEs) Maximum size of structures that are neighbored: VAST+ does not work on MMDB merged structures , because the biological unit of the complete structure is not specified in a computer readable way in the source PDB files. (However, structures that are similar to the individual protein molecules within the merged structure have been calculated by original VAST. To access them, open the MMDB structure summary page for the merged structure of interest and click on the "Similar Structures: VAST+" button near the top of the page. The resulting page will display a note that " no VAST+ neighbors are available ," along with a table showing Original VAST results .) Additional References