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If ATOM entry contains residues other than 20 amino acids (A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y). Then job will terminate abnormally with an error message "Receptor/Ligand Molecule is not in PDB format".


Time profile Example run Flow diagram

Upload PDB files
PROBE is the implementation of an existing algorithm (Flow diagram) by Mitra & Pal (2011). The algorithm has been extensively tested and benchmarked for rigid body molecules. The current version of PROBE will accept bound/unbound protein structures as docking partner. There is no side chain refinement. Please do not upload any NMR file. If you want to dock the structure from NMR file, extract one model and upload that.

Full docking / Scoring and Ranking
The PROBE server can be used for Full docking or for Scoring and Ranking. In case of full docking, the inputs are two protein molecules and few parameters (mentioned under "Full docking"). The server will generate docking decoys using FTDock program module and score them after pruning and refining the structures. On the other hand, if someone wish to score his/her own decoys, then the inputs are two protein molecules and the transformation matrix outputted from docking search to regenerate the decoys. Then the decoys will be scored after refinement. In both the cases, the ranking will be done based on this score value.

Rotation
Default rotational sampling is 12 degree. However, you can go for denser sampling scan by selecting 9 degree rotation or more coarse-grain scan by selecting 15 degree or 18 degree rotation. The accuracy of the result will depend upon the geometry of the protein and the rotation you have selected. If you do not have any idea about it, then we suggest to keep it as default value (12 degree).

convert MSE to MET
The server will remove all the hydrogens and hetero atoms including water except MSE hetero atoms. It will convert MSE hetero atoms to MET (Methionine). So, if you wish to remove MSE hetero atoms, then uncheck the box for converting MSE to MET.

Transformation Matrix
Docking search program outputs a file containing all the information for generation of docking orientations. Upload that file.

For example,
FTDock program outputs ftdock_global.dat (default),
ZDOCK program outputs zdock.out (user defined),
PatchDock program outputs output.txt (user defined),
GrammX program outputs output.res (user defined).

Format of Transformation Matrix
The server will generate the docking orientations from uploaded transformation matrix using uploaded receptor and ligand proteins. Thus, the user is asked to select the correct format of transformation matrix. Currently, PRUNE server supports file format of FTDock, ZDOCK, PatchDock, and GrammX.

DO NOT interchange the receptor and ligand file. Upload only receptor and ligand files along with the transformation file required for generation of docking orientations. If there is need of any intermediate files (like .ms file in PatchDock), that will be generated by the server.

Personal Information
We encourage to provide email id so that job submission and completion notification can be sent through email. Otherwise, you have to bookmark the status page which will appear immediately after job submission. The status page will show the job status information, and after successful completion of the job, will display result. All the jobs in this server are confidential.


Time profile
Figure 1
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Figure 1 indicates time profile of PROBE server. The X-axis indicates the number of residues in receptor and ligand protein molecule and the Y-axis indicates the time requirement in hours. All the parameters (viz, degree of rotation, convert MSE to MET) has kept as default, i.e., we have used 12 degree rotational sampling for docking search. The plot shows that the computation time increases with the increase in protein size.

The computation time has been estimated in a single CPU system of 2.3 GHz processor speed. The figure indicates that docking decoy generation stage is the most time consuming (searching in Figure 1). Thus, to dock large protein molecules, we are suggesting to generate the decoys in your local system and then instead of PROBE server to use our PRUNE server; which takes the list of docking decoys and score the decoys after pruning.

Example run
The example files are at example folder of this server. There are input files (receptor and ligand), PROBEresult file, and predicted complex files (with filename PComplex_N.pdb, where N indicates the rank of the complex). ftdock_trans.txt, along with receptor and ligand files are the input file for scoring and ranking category.

To know how to use PROBE server you can download the receptor and ligand molecule from the example folder and follow the steps as described below.

Step 1:
Upload two protein molecules (number of residues >25) in PDB format as receptor and ligand file (For example, Receptor and Ligand). In order to speed up the computation, it is desireable that larger molecule is selected as receptor molecule. If you wish to dock NMR file, then please upload one model of the NMR file.

Full docking option (Full docking option is checked.)
         Click the radio button before Full docking option.
         The server will eliminate all the hetero atoms except hetero atom MSE. The default option is convert MSE to MET (Methionine) and retain those. If you wish to remove hetero atoms MSE, then uncheck the option to convert it to MET.
         The PROBE server will use FTDock program module to generate docking decoys. The default rotational sampling in FTDock is 12 degree. However, for denser sampling, you have to select lesser degree ot rotation and for more coarse-grain sampling, you have to select higher degree of rotation. The computation time increases with the decrease in rotation, so, user is requested to properly utilize this option. In case, you do not familiar with this option, user is suggested to keep the default.
         User is requsted to mention the the number of ranked complexes (s)he wants. The default value is 10.

Scoring and Ranking option (Scoring and Ranking option is checked.)
         Click the radio button before Scoring and Ranking option.
         Upload the transformation matrix ftdock_trans.txt which is generated by docking search FTDock. So, select FTDock under "Format of Transformation Matrix".

The time requirement for docking (see Time profile) varies based on the size of the proteins, the parameters you have selected and the load of the server. Thus, facility is provided to sent job submission and job completion notification through email. However, this is an optional information.

Figure 2
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Step 2:
Full docking option (Full docking option is checked.)
         The PDB files will be preprocessed after successful upload. First, it will eliminate all the hydrogens, waters and non-proteinous atoms. If there exists multiple conformation for an atom/residue, then the first one will be selected and the rest will be discarded. After that, the server will show a brief summary of the proteins (Figure 3).
         If you find everything is fine, press the button "Start PROBEing".

Scoring and Ranking option (Scoring and Ranking option is checked.)
         The server will show a brief summary of the proteins. Press the button "PROBE score".

The job will be submitted at the linux cluster and the job submission notification, with the link of the status of the job, will be sent through email. In case, email id is not provided, you have to bookmark the status page. All the job status related information including result will appear in the status page, which appears just after the submission of the job.

Figure 3
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Step 3:
Job completion notification, with the link of the result page, will be sent through another email (if email id is provided). The result page will contain the following information:

Modified receptor and ligand file: During preprocessing stage, the PDB file will be modified (removal of hydrogens, waters and non-proteinous atoms; conversion of MSE to MET; removal of multiple conformations). Those modified files are made available for download from result page.

PROBE result file: The parameters of each decoys (complexes) are tabulated in the PROBE result page. User can download this file.

Tabulated result: The detailed result is also tabulated in the result page. The first column is hyper linked to the complex structure; which can be downloaded from PROBE server. The facility is provided to visualize the complex structure without downloading the complex. To visualize the complex click on Jmol view option. The complex structure will be dispayed at a new tab of your browser. There is no need of any visualization software installed in your system. The only requirement is java runtime environment so that your browser can run java applet.

Download all complexes: A separte link is provided at the end of the result table (not shown in figure) to download all the predicted complexes as a single .tar.gz file in a single click.

Figure 4
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Flow diagram
The flow diagram of PROBE is shown in Figure 5. The procedure is divided into three stages, represented by three different background colors.

In the first stage (pink background) FTDock program's search module is used to generate docking decoys. Grid size and angle step determines the number of iterations (say N) made to generate docking decoys in FTDock. At each iteration, best 3 decoys are being retained. This gives a total of 3N number of decoys, which are screened at the second stage.

In the second stage (white background), the complexes are screened based on the distribution of the IA and/or NE and SE.

In the third stage (cyan background), grouping, scoring and ranking is done.

For "Full docking", although all the three steps will be executed, but for "Scoring and Ranking", since user is supplying docking decoys, so first step will not be executed.

Figure 5
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Happy PROBEing !!!
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