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Tutorial: 3D motif search

In this example, we will design a 3D motif from the active site of 1dmt, a neutral endopeptidase and search for structures containing a similar arrangement of residues.

Step by Step

  • First of all, open the pdb file 1dmt (using the File:Import menu)
  • Using the Control Panel, Select the Zinc atom (Zn755) .
  • Using the Select menu, Select Neighbors of Selected Residue (cutoff 4.5A)
  • Click on the 'return' key to show only those residues, and center the view clicking one the top left icon of the toolbar.

At this stage, you should be in this situation.

3Dsearch1

  • Now using the distance measuring tool, measure a few key distances that will be used as constaints for the search so as to obtain this:

3Dsearch2

  • Now use the Tool Menu: Generate 3D motif from selection to write a 3D motif. The motif is automatically generated from the selected rresidues, and includes residues for which distance constraints have been generated.
  • You should obtain something like this:
    3D search 3b
  • As the motif is rather stringent, we will alter it to relax the constraints. Of course some thinking should be put in how to relax the constraints so as to remain selective and also cut the search time. The more stringent, the faster.
  • In our case, we will want to remain specifcic for Histidines (group 0,1 and 3) but we might want to relax the constraint on the Gutamic acid to allow an Aspartic acid. Also it might be appropriate to lift the secondary structure constraints. Finally, we will also remove most of the backbone separation constraints, and keep just one (although fairly permissive) between the two first Histidines of the motif. By default, the minimal and maximal range of each distance constraint is set to be exactly 0.5 Angstrom less and more than the measured distance. This might be too restrictive in some situations, and here we will want to augment the maximum tolerated distance to 1A.
  • You should obtain something like this: (right click on the link to save it to your local disk).
#SEARCH3D # pattern defined from: 1dmt # list of residues # GroupNum allowed_kind allowed_Sec_Struct ; name chain num GROUP 0 H * ; 'HIS' 'A' '583 ' GROUP 1 H * ; 'HIS' 'A' '587 ' GROUP 2 ED * ; 'GLU' 'A' '646 ' GROUP 3 H * ; 'HIS' 'A' '711 ' ATMTP 4 Zn * ; ' ZN' 'A' '755 ' 'ZN ' # distances constraints # (FromGrp FromAtom ToGrp ToAtom minDist optimalDist maxDist) DIST 1 CG 0 CG 5.1 5.6 6.6 DIST 1 CG 3 CG 8.8 9.3 10.3 DIST 1 CG 4 Zn 3.7 4.2 5.2 DIST 2 CB 1 CG 6.6 7.1 8.1 DIST 2 CB 3 CG 4.0 4.5 5.5 DIST 3 CG 0 CG 7.6 8.1 9.1 # backbone separation # (FromGrp ToGrp min max) DELTA 1 0 1 75 # END
  • Now go back to Swiss-PdbViewer and clear all labels (Display:Labels:Clear User Labels).
  • Deselect all residues (Select:None).
  • Go to the Tool Menu: Search 3D motif in current layer and select the motif file you just edited.
  • It should open up in a text window, and you will notice that a new line starting with 'Found 1dmt residues:' has been added at the bottom.
  • Click on that line. The motif should now be highlighted, and residues participating in the motif should be selected.
  • Now Select:all, Edit:Blast selection against ExPDB to find other structures similar to 1dmt.
  • It should identify 1y8j. Click on the blue hyperlink to load the structure in the workspace. At this point the two structures are not superposed in 3D space.
  • Using the Control Panel, make the 1dmt layer active.
  • Click once more on the search3D motif results line 'Found 1dmt residues:' so as to ensure that only the residues particiapting in the motif are selected.
  • Now make 1y8j the active layer.
  • Go to the Tool Menu: Search 3D motif in current layer and select the edited motif file we used so far.
  • It should open up in a text window, and you will notice that a new line starting with 'Found 1y8j residues:' has been added at the bottom.
  • Click on that line. The motif should now be highlighted, and residues participating in the motif should be selected. Furthermore, 1y8j should now be superposed onto 1dmt, in 3D space, based on the residues participating in the motif!
  • The blast has not identified any distantly related structures. However, we can now use our motif to look for other structures containing this motif.
  • Use the Tool:Submit 3D motif search in subset of PDB. This will submit the request to the vital-it server and scan a non-redundant subset of structures to identify which ones contain this motif.
  • The server will return a unique ID that can be used to access the results from the File:Import menu. Results are kept for one week on the server.
  • The unique ID is also added to the list of submitted jobs for the current session (in the Swissmodel:Check status of submitted jobs submenu). By default, Swiss-PdbViewer will automatically display the results once they become available. However, note that as results are displayed as soon as at least one hit is identified, this might lead to display incomplete results. In that case, the Swissmodel:Check status of submitted jobs menu can be used to refresh the view and check if complete results are availaible.
  • In our example, you should get something like this:
#SEARCH3D # pattern defined from: 1dmt # list of residues # GroupNum allowed_kind allowed_Sec_Struct ; name chain num GROUP 0 H * ; 'HIS' 'A' '583 ' GROUP 1 H * ; 'HIS' 'A' '587 ' GROUP 2 ED * ; 'GLU' 'A' '646 ' GROUP 3 H * ; 'HIS' 'A' '711 ' ATMTP 4 Zn * ; ' ZN' 'A' '755 ' 'ZN ' # distances constraints # (FromGrp FromAtom ToGrp ToAtom minDist optimalDist maxDist) DIST 1 CG 0 CG 5.1 5.6 6.6 DIST 1 CG 3 CG 8.8 9.3 10.3 DIST 1 CG 4 Zn 3.7 4.2 5.2 DIST 2 CB 1 CG 6.6 7.1 8.1 DIST 2 CB 3 CG 4.0 4.5 5.5 DIST 3 CG 0 CG 7.6 8.1 9.1 # backbone separation # (FromGrp ToGrp min max) DELTA 1 0 1 75 # END --------------------------------------------------------------------------------------- Results (if any) will appear below, one hit per line. Open this result file as a text file with Swiss-PdbViewer to visualize the hits. Clicking on a hit will check if a layer with the corresponding name has been loaded if this is the case, it will select the residues and highlight the contstraints if this is not the case, it will prompt you whether you want to download the pdb file Note that clicking on the hit while the Control key is pressed will always result in loading the pdb file in a new layer, which could be useful to compare several hits from the same pdb entry. --------------------------------------------------------------------------------------- @2IW0;HIS.A.104 ;HIS.A.108 ;ASP.A.49 ;HIS.A.206 ; ZN.A.1255.0|HHD|scscc @8TLN;HIS.E.142 ;HIS.E.146 ;GLU.E.166 ;HIS.E.231 ; ZN. .321 .0|HHE|hhhcc @1R1H;HIS.A.583 ;HIS.A.587 ;GLU.A.646 ;HIS.A.711 ; ZN.A.1001.0|HHE|hhhcc @1U4G;HIS.A.140 ;HIS.A.144 ;GLU.A.164 ;HIS.A.223 ; ZN. .9800.0|HHE|hhhcc @2CC0;HIS.A.62 ;HIS.A.66 ;ASP.A.12 ;HIS.A.155 ; ZN.A.200 .0|HHD|scssc @2CC0;HIS.B.62 ;HIS.B.66 ;ASP.B.12 ;HIS.B.155 ; ZN.B.200 .0|HHD|scssc --------------------------------------------------------------------------------------- part| status | search | hits | progress log: 1| done| ok| 0| progress log: 2| done| ok| 2| progress log: 3| done| ok| 2| progress log: 4| done| ok| 2| Note: for a list of the pdb entries searched, go to http://spdbv.vital-it.ch/currentSearch3Dset.txt if your favorite file is not in this list, the search has to be done on your Mac/PC using the Search 3D motif in current layer option from the Tool Menu.
  • Click on the result line starting with '@1U4G;'
  • The motif should now be highlighted, and residues participating in the motif should be selected. Furthermore, the pseudomonas elastase 1U4G should now be superposed onto the human neprylisin 1dmt, in 3D space, based on the residues participating in the motif.
  • Now, make the layer 1U4G the active one.
  • Use the Fit:Generate Structural Alignment menu
  • Color: RMS
  • As you can see in the Alignment Window (Wind:Alignment), a few key residues of the active site are conserved despite the very low percent identity between those two structures.
  • You can use the Select:residues whose RMSD to reference structure is below 3.0 Angstroms to further explore the active site conservation.

3Dsearch4b

Finally, here are two other examples of motifs that might serve as a starting point to build your own motifs:

  • The first one looks for leucine zippers and has been defined from the pdb entry 1ce9 Leucine Zipper: (right click on the link to save it to your local disk).
  • The second one looks for proteins that contain pyrophosphate (which could be a single group, or part of ATP for example) it has been defined from the pdb entry 1atp using a tolerance distance of +/- 20% of the bond length. PPI: (right click on the link to save it to your local disk).