Hemochromatosis: Sequence based predictions

Secondary Structure

In this task, secondary structure of proteins is predicted using the programs reProf and PsiPred. The results are then compared to the DSSP[1] secondary structure assignments from corresponding crystal structures in the PDB. Thus, the following crystal structures were selected for comparison. The first priority for selection was to get the protein in its native state and not bound to another molecule. The other criteria were the quality of the structure and the alignment to the protein sequence(having one continous segment).

Uniprot			PDB
UID	Name	Length	ID	Resolution [A]	Chain	Length
Q30201	Hereditary hemochromatosis protein	348	A16Z	2.60	A	275
P10775	Ribonuclease inhibitor	456	2BNH	2.30	A	457
Q9X0E6	Divalent-cation tolerance protein CutA	101	1VHF	1.54	A	113
Q08209	CAM-PRP catalytic subunit	521	1M63	2.80	A	372

In the next step, the output of the prediction programs and the DSSP assignments have to be made comparable. DSSP assigns 8 different classes of secondary structure, whereas reProf and PsiPred only predict helix(H), sheet(E) and loop(L or C). Therefore, H and G are mapped to H, E to E and all other DSSP classes to C.

To assess the quality of the prediction, the class specific accuracy, coverage and F1-measure were used along with the Q3 and SOV3<ref>Zemla. A, et al.;A Modified Definition of Sov, a Segment-Based Measure for Protein Secondary Structure Prediction Assessment; PROTEINS 34:220–223 (1999)</ref> measures. The Q3 is defined as follows:
<math> Q_3 = \frac{\text{correct predictions}}{ \text{all predictions}} = \frac{TP + TN}{TP + FP + TN + FN } } </math>

Reprof takes as input either sequences or PSSMs. Therefore PSSMs were generated by querying the HFE(Q30201) sequence against the big_80, SwissProt and PDB databases. The tool of choice for this was PsiBlast with standard parameters(2 iterations and e-value cutoff of 0.002).

Prediction methods
	Reprof + Sequence			Reprof + Big80			Reprof + SwissProt			Reprof + PDB			Psipred
Q3	0.76			0.81			0.86			0.84			0.84
SOV3	0.66			0.75			0.84			0.84			0.73
	Acc	Cov	F1	Acc	Cov	F1	Acc	Cov	F1	Acc	Cov	F1	Acc	Cov	F1
H	0.63	0.33	0.44	0.74	0.48	0.59	0.84	0.65	0.74	0.85	0.52	0.64	0.98	0.77	0.86
C	0.57	0.69	0.63	0.63	0.75	0.68	0.68	0.81	0.74	0.64	0.83	0.72	0.61	0.95	0.74
E	0.79	0.63	0.70	0.81	0.84	0.83	0.89	0.86	0.87	0.88	0.85	0.86	0.94	0.55	0.69

The deciding criteria for the reprof method choice were the Q3, SOV3 and F1-measures. For the Q3 and the SOV3, the SwissProt PSSM performed best and the F1-measures were also reasonable. Therefore, this method was chosen for all future predictions.

Protein	Method	Q3	SOV3
Q08209	ReProf	0.83	0.78
Q08209	Psipred	0.87	0.79
Q30201	ReProf	0.86	0.84
Q30201	Psipred	0.84	0.73
P10775	ReProf	0.91	0.93
P10775	Psipred	0.93	0.94
Q9X0E6	ReProf	0.75	0.65
Q9X0E6	Psipred	0.89	0.86

Hemochromatosis SS Alignments

While SOV3 and Q3 do not always agree which of the two methods performs better, they do so most of the time. Nevertheless, it was decided that SOV3 is to be trusted more than Q3, because it takes into account the per segment accuracy rather than just the per residue accuracy. Thus, PsiPred outperforms reprof in 3 out of 4 cases. It is also notable, that the SOV3 values range from 0.65 for Q9X0E6(101 residues) to 0.94 for P10775(456 residues). So the performance of the method depends on the length of the protein and the protein itself, but PsiPred performed best overall.

Disorder

UID	DisProt ID	SeqID	E-value	Method
Q08209	DP00092	-	-	direct match
Q30201	DP00670	0.29	2e-30	psiblast
P10775	DP00465	0.4	7e-30	psiblast
Q9X0E6	DP00564	0.25	0.36	smith-waterman

<figure id="Disorder_plot_Q30201"></figure>	<figure id="hemo_plot_P10775"></figure>
<figure id="hemo_plot_Q9X0E6"></figure>	<figure id="hemo_plot_Q08209"></figure>

Transmembrane Helices

Uniprot			PDB
UID	Name	Length	ID	Resolution [A]	Chain	Length
Q30201	Hereditary hemochromatosis protein	348	A16Z	2.60	A	275
Q9YDF8	Voltage-gated potassium channel	295	1ORQ	3.20	C	223
P35462	D(3) dopamine receptor	400	3PBL	2.89	A	481
P47863	Aquaporin-4	323	2D57	3.20	A	301