View source for Gaucher Disease: Task 03 - Lab Journal

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== Secondary structure ==

'''ReProf''' uses a fasta sequence or a PSI-BLAST PSSM for prediction, '''PsiPred''' a fasta sequence and '''DSSP''' server needs a PDB file in order to use the 3D coordinates of atoms. The predictions were made for the four proteins, including the Gaucher's disease-causing protein (<xr id="secondary structure proteins"/>). If several PDB structures are available, the one covering the most UniProt sequence most similarly was chosen. For glucosylceramidase the structure 1OGS was used (as in the task 2).

<figtable id="secondary structure proteins">
{| class="colBasic2"
! colspan="4" | Uniprot
! colspan="5" | PDB
|-
! Entry || Protein name || Origin || Length || Entry || Method || Resolution (Å) || Chain || Positions
|-
|[http://www.uniprot.org/uniprot/P10775 P10775] || Ribonuclease inhibitor || pig || 456 || [http://www.pdb.org/pdb/explore/explore.do?pdbId=2BNH 2BNH] || X-ray || 2.30 || A || 1-456
|-
|[http://www.uniprot.org/uniprot/Q9X0E6 Q9X0E6] || Divalent-cation tolerance protein CutA || bacterium Thermotoga maritima || 101 || [http://www.pdb.org/pdb/explore/explore.do?pdbId=1VHF 1VHF] || X-ray || 1.54 || A || 2-101
|-
|[http://www.uniprot.org/uniprot/Q08209 Q08209] || Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform, EC=3.1.3.16 || human || 521 || [http://www.pdb.org/pdb/explore/explore.do?pdbId=1AUI 1AUI] || X-ray || 2.10 || A || 1-521
|-
|[http://www.uniprot.org/uniprot/P04062 P04062] || Glucosylceramidase/acid-beta-glucosidase, EC=3.2.1.45 || human || 536 || [http://www.pdb.org/pdb/explore/explore.do?pdbId=1OGS 1OGS] || X-ray || 2.00 || A/B || 40-536
|-
|}
<center><small>'''<caption>''' The four UniProt protein sequences and corresponding PDB structures selected for the secondary structure prediction with ReProf and PsiPred and assignment with DSSP.</caption></small></center>
</figtable>

The script of the Phenylketonuria group [[Phenylketonuria/Task3/Scripts | filter_secStruc.pl]] was used to extract the secondary structures in the three letter code: E, H and L (<xr id="secondary structure code"/>). For DSSP irregular regions are encoded as "-". Then, precision for each one of the three secondary structures and in total, ignoring positions without a secondary structure ("-"), was calculated using the second script of the Phenylketonuria group, [[Phenylketonuria/Task3/Scripts | SecStrucComparison.jar]] as follows:

<code><center>precision = number of matches / number of residues </center></code>

<figtable id="secondary structure code">
{| class="colBasic2"
|-
! colspan="4" | "Secondary Structure Code" 
|-
! Secondary structure || ReProf || PsiPred || DSSP
|-
| Helix (alpha) || H || H || GHI
|-
| Extended strand (beta) || E || E || BE
|-
| Loops/Turns || L || C || ST
|-
|}
<center><small>'''<caption>''' The different types to represent secondary structure in ReProf, PsiPred and DSSP.</caption></small></center>
</figtable>

First different PSSMs after different PSI-BLAST runs (all combinations: against big_80/swissprot database, 2/3 iterations, E-value 2E-3/10E-10/10E-20) were tested on the shortest protein, Q9X0E6, then the run parameters yielding the best precision compared to PsiPred and DSSP were chosen. The best parameters were: '''big_80, 3 iterations and evalue cutoff 10E-10''', which were then applied to create PSSMs for the other proteins. (The table where the results for all parameters are summarized can be seen here: <code>/mnt/home/student/kalemanovm/master_practical/Assignment3_SequenceBasedPredictions/SecondaryStructure/reprof_out/parsedSecStr/README.Q9X0E6.psiblast_param.precision</code>.)

Other used scripts and created data can be found in: /mnt/home/student/kalemanovm/master_practical/Assignment3_SequenceBasedPredictions/SecondaryStructure.

== Disorder ==
'''IUPred'''
We called IUPred with "long", "short" and "glob" modes the same four proteins (P10775, Q08209, Q9X0E6, P04062). One can generate graphical output with the [http://iupred.enzim.hu/ IUPred server]. However, for a clear view and interpretation of the results of the three options, we combined them in a single plot with an R script. 


'''MD (MetaDisorder)'''
The output files ending on ".md.out.mdisorder" have the following columns (example for P10775):

 Number Residue NORSnet NORS2st PROFbval bval2st Ucon Ucon2st MD_raw   MD_rel  MD2st
     1   M       0.19    -       0.99    D       0.15    -       0.551   1       D
     2   N       0.11    -       0.78    D       0.21    -       0.475   1       -
     3   L       0.08    -       0.65    D       0.26    -       0.455   2       -
     4   D       0.13    -       0.68    D       0.20    -       0.414   3       -
     5   I       0.09    -       0.40    -       0.22    -       0.364   5       -
     6   H       0.14    -       0.48    D       0.29    -       0.384   4       -
     7   C       0.58    D       0.57    D       0.26    -       0.384   4       -

''Key for output:''
Number - residue number;
Residue - amino-acid type;
NORSnet - raw score by NORSnet (prediction of unstructured loops);
NORS2st - two-state prediction by NORSnet; D=disordered;
PROFbval - raw score by PROFbval (prediction of residue flexibility from sequence);
Bval2st - two-state prediction by PROFbval;
Ucon - raw score by Ucon (prediction of protein disorder using predicted internal contacts);
Ucon2st - two-state prediction by Ucon;
MD - raw score by MD (prediction of protein disorder using orthogonal sources);
MD_rel - reliability of the prediction by MD; values range from 0-9. 9=strong prediction;
MD2st - two-state prediction by MD.

NORSnet, PROFbval and Ucon raw scores are the predicted tendencies of those methods and MD_raw is the final raw score by MD. In all 2-state scores, a residue is predicted to be disordered (D), if the raw score is higher than 0.5. We wrote an R script to plot all the raw scores for each protein.

All the used scripts and created data can be found in: /mnt/home/student/kalemanovm/master_practical/Assignment3_SequenceBasedPredictions/Disorder.

==Transmembrane Helices==
The Memsat-SVM predictions were processed on the web server [http://bioinf.cs.ucl.ac.uk/psipred/ Psipred]. To get the membrane helices prediction I chose the option MEMSAT3 & MEMSAT-SVM instead of the default option.

Polyphobius was run with the script provided in the subsection [https://i12r-studfilesrv.informatik.tu-muenchen.de/wiki/index.php/Resource_software Software]. It can also be found on the rostlab server <code>/mnt/home/student/gerkej/gaucher/task3/tmh/polyphobius.pl</code>. It automatically includes <code>Blast</code> and <code>kalign</code>

==Signal Peptides==
To find out more about signal peptides the following webservers were used:
* [http://www.cbs.dtu.dk/services/SignalP/ SignalP]: version 4.1
* [http://www.signalpeptide.de/index.php?m=listspdb_mammalia Signal Peptide Database]: last update, 6th November 2010

On the Signal Peptide Database we also had a look on the transmembrane helices for the three proteins analyses in this exercise. Except of Glucocerbrosidase which does not have a documented membrane helix in the SP Database, the other three proteins of the "Membrane Helices" exercise are not listed in the Signal Peptide Database.

==GO Terms==
The GO Terms were reached by using the corresponding webservers of 

*[http://genius.embnet.dkfz-heidelberg.de/menu/cgi-bin/w2h-open/w2h.open/w2h.startthis?SIMGO=w2h.welcome GOPET]
*[http://www.cbs.dtu.dk/services/ProtFun/ ProtFun 2.2 Server]
*[http://pp-dev.informatik.tu-muenchen.de:8080/home PredictProtein]
*[http://pfam.sanger.ac.uk/ Pfam 27.0] , last update, March 2013