Sequence-based predictions (PKU)

In this task we will find out, how much information one can get from the plain sequence of our disease. We try to pretend we know nothing but the freshly sequenced primary-structure. We will perform predictions which are available as a web service as well as local programs to determine the functions and structure of out disease. Afterwards we will analyse the results with our prior knowledge about Phenylketonuria. In this manner we hope to get an idea how reliable the information derived from such tools is.

Short Task Description

Our task is to use the primary sequence of our protein (and some other example proteins) to predict comparatively simple features like secondary structure, signal peptides and transmembrane regions and more advanced like GO terms and similar functional annotations with different tools. The used commands and programms are listed at the appropriate places (if short and interesting enough) or linked at their own site. For a more detailed instruction please read.. and while you're at it, read some more

Secondary Structure Prediction

In this section we show a comparison of several secondary-structure-prediction-tools. In order to have the gold standard we will analyse the results with the information from DSSP.<ref name=DSSP> Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, 22, 2577-2637.</ref>

Test some SS-predictors ans compare the results to the 'gold standard' of dssp

ReProfSeq

reprof -i <uniprotID>.fasta
egrep -v "^#|^No" <uniprotID>.reprof |awk '{print $3}'|tr -d '\n' > <uniprotID>_reprof.secstruc

PsiPred

Webserver here

grep Pred: <uniprotID>.psipred_out |cut -d " " -f2|tr -d '\n' > <uniprotID>_psipred.secstruc

DSSP

Download dssp here and get PDB files matching the Uniprot entries

dssp -i <PDBID>.pdb > <PDBID>.dssp
tail -n+29 <PDBID>.dssp |cut -c17|tr ' ' '-'|tr -d '\n' > <PDBID>_dssp.secstruc

PDB-Files contains only part of the structure or more than 1 chain! e.g. 117aa-424aa for PAH.

Aligned structure to sequence manually..

Script to calculate Q3 and SOV: ss_score.py

results

Protein	ReprofSeq
UniprotID	Q_E	Q_H	Q_L	Q_3	SOV_E	SOV_H	SOV_L	SOV
P00439	45.5	75.7	74.8	71.2	47.5	86.7	71.8	76.4
P10775	23.1	71.9	62.0	61.8	21.9	87.2	66.9	70.6
Q9X0E6	26.8	91.9	39.1	53.5	36.1	91.1	41.0	57.6
Q08209	69.1	34.0	73.1	57.9	62.4	45.6	76.7	63.0

Protein	PsiPred
UniprotID	Q_E	Q_H	Q_L	Q_3	SOV_E	SOV_H	SOV_L	SOV
P00439	57.6	85.1	89.0	83.8	57.6	88.5	54.4	71.1
P10775	92.3	90.3	94.7	92.5	92.7	95.5	95.7	95.2
Q9X0E6	75.6	86.5	78.3	80.2	93.44	100.0	94.5	96.1
Q08209	58.2	77.3	90.7	81.0	64.1	85.8	64.8	72.5

Signalpeptides

P47863 from rat, rest human => type eukaryotes; command:

signalp -trunc 70 -t euk <ID>.fasta > <ID>.sigP_out

	SignalP v3			SignalP v4	Signal Peptide Database
UniprotID	SignalP-HMM	SignalP_NN	summary prediction	summary prediction	experimental
P00439	0% signal peptide or anchor	5 times No	no signal peptide or anchor	no signal peptide or anchor	no evidence for peptide or anchor
P02768	100% signal peptide, 0% signal anchor	5 times Yes	signal peptide	signal peptide	confirmed signal peptide
P11279	100% signal peptide, 0% signal anchor	5 times Yes	signal peptide	signal peptide	confirmed signal peptide
P47863	52.6% signal peptide, 45.7 signal anchor	4 No, 1 Yes	signal peptide	no signal peptide or anchor	no evidence for peptide or anchor

P47863 reaches a high maximal signal peptide score (S-score) in signalP-NN and probabilities above the cut-off in signalP-HMM in version 3. In version 4 (webserver) neither version predicts a signal peptide for this protein and the probabilities/scores are well below the cut-off values. P47863 is a multipass membrane protein and signalPv3 most likely predicted a possible cleavage site for the N-terminal membrane region, classifying it wrongly as signal sequence.

GO Terms

GOPet

GOPet predicted GO terms
GO ID	Aspect	Confidence	GO Term	True/False
GO:0003824	F	94%	catalytic activity	true
GO:0016491	F	88%	oxidoreductase activity	true
GO:0004497	F	87%	monooxygenase activity	true
GO:0004505	F	84%	phenylalanine 4-monooxygenase activity	true
GO:0004510	F	80%	tryptophan 5-monooxygenase activity	false
GO:0004511	F	79%	tyrosine 3-monooxygenase activity	false
GO:0046872	F	78%	metal ion binding	true
GO:0005506	F	78%	iron ion binding	true
GO:0008199	F	72%	ferric iron binding	false
GO:0008198	F	72%	ferrous iron binding	false
GO:0016597	F	71%	amino acid binding	true

ProtFun

# Functional category                  Prob     Odds
 Amino_acid_biosynthesis           => 0.210    9.530
 Biosynthesis_of_cofactors            0.229    3.180
 Cell_envelope                        0.034    0.563
 Cellular_processes                   0.063    0.867
 Central_intermediary_metabolism      0.061    0.970
 Energy_metabolism                    0.343    3.815
 Fatty_acid_metabolism                0.025    1.889
 Purines_and_pyrimidines              0.392    1.615
 Regulatory_functions                 0.020    0.125
 Replication_and_transcription        0.118    0.438
 Translation                          0.204    4.630
 Transport_and_binding                0.024    0.060

# Enzyme/nonenzyme                     Prob     Odds
 Enzyme                            => 0.724    2.527
 Nonenzyme                            0.276    0.387

# Enzyme class                         Prob     Odds
 Oxidoreductase (EC 1.-.-.-)          0.154    0.738
 Transferase    (EC 2.-.-.-)          0.271    0.785
 Hydrolase      (EC 3.-.-.-)          0.083    0.261
 Lyase          (EC 4.-.-.-)          0.047    1.002
 Isomerase      (EC 5.-.-.-)       => 0.100    3.138
 Ligase         (EC 6.-.-.-)          0.019    0.370

# Gene Ontology category               Prob     Odds
 Signal_transducer                    0.075    0.350
 Receptor                             0.003    0.016
 Hormone                              0.001    0.206
 Structural_protein                   0.005    0.166
 Transporter                          0.025    0.229
 Ion_channel                          0.010    0.168
 Voltage-gated_ion_channel            0.005    0.232
 Cation_channel                       0.010    0.215
 Transcription                        0.043    0.334
 Transcription_regulation             0.032    0.255
 Stress_response                      0.010    0.118
 Immune_response                      0.012    0.140
 Growth_factor                        0.006    0.407
 Metal_ion_transport                  0.009    0.020

Additional Predictions:

Feature 	Output summary
   SignalP 3.0 	  No signal peptide cleavage site predicted 
   ProP 1.0 	  1 propeptide cleavage site predicted at position:   74 
   TargetP 1.1 	  No high confidence targeting predition 
   NetPhos 2.0 	  22 putative phosphorylation sites at positions 16 23 40 70 110 196 250 303 339 391 411 22 105 189 278 418 24 77 198 268 277 317
   NetOGlyc 3.1   No O-glycosylated sites predicted
   NetNGlyc 1.0   2 putative N-glycosylated sites at positions 61 376
   TMHMM 2.0 	  No TM helices predicted

References

<references/>