Canavan Disease: Task 06 - Protein Structure Prediction

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LabJournal

Dataset

To gain the HRas multiple sequence alignment the instructions were followed and the full MSA provided by Pfam (PF00071) was downloaded and used for further calculations and statistics. Searching for a multiple sequence alignment for ASPA/ACY2 in Pfam revealed that the two criteria to gain meaningful insights out of the calculations of freecontact, evcouplings and evfold, namely over 1000 sequences in the MSA and large parts of the reference sequence are contained in the MSA, are satisfied. The multiple sequence alignment for the protein family containing ASPA (PF04952) includes 2822 sequences and the region of ASPA that is used in the MSA spans from position 10 to 301 with ASPA having a total length of 313 amino acids. Hence the Pfam MSA is regarded as viable input for the following calculations.

HRAS

Freeconact is based upon searching conserved regions and correlated mutations in a multiple sequence alignment, to predict pairs of residues that are in contact in a protein. It is to be expected that residues that are close to each other in sequence are as well close in three dimensional space, as their contact often defines the secondary structure elements and the conformation of the protein on a small scale. Therefore residue pairs that are close in sequence are ranked with a high CN-score by freecontact. However more meaning full for the overall conformation of the protein are stabilizing contacts between residues that are more distant in sequence space. This is the reason why filtering the predicted contacts to exclude residues that are distant more than five residues in sequence. Looking at the distribution of the cn-scores (<xr id="hras_cn_distribution">Figure </xr>) this gets visible as well.

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<figure id="hras_cn_distribution">

The distribution of the cn-scores for HRAS calculated by freecontact. The frequency of the cn-scores are displayed for all pairs of residues (orange) and pairs of residues more than five positions apart in the sequence (blue). Pairs with a cn-score of above 1 are considered high scoring. It is visible that only a tiny fraction of the pairs are high scoring, as well as the reduction of the set to pairs of a sequence distance of more than five has a huge impact of the amount of high scoring pairs.

<figure id="hras_freecontact_contactmap">

Contact map of HRAS (121P) calculated form the crystal structure. Displayed in grey are the residue pairs with a distance of less than 5Å. Displayed as red dots are the contacts predicted by freecontact. Those are reduced to residue pairs that have a high cn-score (cn > 1) and are more than 5 residues apart in sequence (i & i+n, where n > 5). The dashed rectangle in green visualizes the borders in which freecontact calculated cn-scores (residue 5 to 165).

The first thing to be noted is that only a tiny fraction (514 out of 12561 possible pairs) has a cn-score > 1, what is considered to be high scoring. If the set is reduced to residue pairs with a sequence distance greater five this subset of high scoring pairs is imideately reduced to 65 pairs. Secondly the maximal cn-scores is reduced from 6.01 to 3.40. Reducing the set however has no great impact on the precision. The predicted high scoring contacts of the orginal set contain 439 true positives and 75 false positives (precision of 0.854) while the reduced set contains 55 true positive predictions out of 65 predictions over all (precision of 0.846). The predicted contacts are visualized together with the actual contacts calculated with the aid of the crystal structure in <xr id="hras_freecontact_contactmap"> Figure </xr>. A overview of the top 20 predictions for HRAS in more detail are displayed in <xr id="top_20_hras"> Table </xr>.

<figtable id="top_20_hras">

Predicted residue contacts for HRAS by freecontact
Residue #1 Residue #2 CN-Score TP/FP
Position Amino acid Position Amino acid
11 A 92 D 3.40454 TP
81 V 116 N 2.99937 TP
87 T 129 Q 2.68523 FP
82 F 141 Y 2.52755 TP
84 I 115 G 2.52502 TP
19 L 81 V 2.50464 TP
82 F 115 G 2.41709 TP
10 G 16 K 2.26384 TP
130 A 141 Y 2.24938 TP
123 R 143 E 2.21315 TP
114 V 155 A 2.09971 TP
17 S 57 D 1.98068 TP
80 C 93 I 1.96126 TP
142 I 158 T 1.90419 TP
78 F 100 I 1.82996 TP
25 Q 40 Y 1.66144 TP
116 N 146 A 1.63396 TP
8 V 20 T 1.60439 TP
72 M 99 Q 1.59088 TP
118 C 150 Q 1.51682 TP
Overview of the top 20 residue pairs predicted to be in contact that are apart more than 5 residues in the sequence (i & i+n, where n > 5). The residue pairs are ranked in descending order according to their cn-score calculated by freecontact. Of those 20 residue pairs only one (87T->129Q) has no actual contact when compared to the crystal structure.

</figtable>

ASPA

Tasks

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