Canavan Disease: Task 06 - Protein Structure Prediction

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

Calculation and analysis of correlated mutations

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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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.

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).

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<figure id="hras_freecontact_contactmap">

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 10 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

Predicted residue contacts for HRAS by EVcouplings Residue #1 Residue #2 CN score TP/FP Position Amino acid Position Amino acid 10 G 16 K 0.2058040 TP 13 G 21 I 0.1126380 FP 11 A 92 D 0.0946234 TP 87 T 129 Q 0.0766384 FP 114 V 155 A 0.0760636 TP 117 K 145 S 0.0758184 TP 82 F 141 Y 0.0757755 TP 116 N 146 A 0.0755790 TP 35 T 60 G 0.0707763 TP 81 V 116 N 0.0665576 TP

Overview of the top 10 residue pairs predicted to be in contact by freecontact and EVcouplings 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/DI score calculated by freecontact/EVcouplings. Of the 10 residue pairs calculated by freecontact only one (Thr 87 -> Gln 129) has no actual contact when compared to the crystal structure. Within the top 10 residue pairs calculated by EVcouplings two are false positive. Gly 13 -> Ile 21 and interestingly Thr 87 -> Gln 129, the pair miss predicted as well by freecontact.

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Searching for evolutionary hotspots, the L best high-scoring residue couplings with a sequence distance greater than five, where L is the length of the aligned sequence in the multiple sequence alignment were extracted. In the case of HRAS freecontact used a sequence part of length 160 to create the couplings. The CN scores of these 160 couplings are then summed up for each amino acid. If these sums are normalized (dividing the sums by 160) they can give hints how evolutionary important the amino acid is. Performing this procedure resulted in the observation that for HRAS Phe 82, Val 81, Tyr 141, Glu 143 and Gly 115 (in descending order) seem to be the evolutionary most important residues in terms of forming and stabilizing the protein.

A further possibility to predict contacts apart from freecontact is using EVcouplings. The results EVcouplings delivers are filtered the same way as the freecontact results. All scores for residue pairs that are less than five sequential positions apart are excluded. The remaining couplings are sorted after their DI score (a former version of the CN score). Comparing the top 50 DI scores from EVcouplings and CN scores from freecontact an overlap of 20 couplings can be observed. However within the 10 best pairs of each method, there is an overlap of five pairs, namely Gly 10 -> Lys 16, Ala 11 -> Asp 92, Thr 87 -> Gln 129, Phe 82 -> Tyr 141, Val 81-> Asn 116. Interestingly one of the residue pairs (Thr 87 -> Gln 129), that is predicted by both methods is a false positive. A more detailed view of the top 10 ranked residue pairs calculated by EVcouplings can been seen in <xr id="top_20_hras"> Table </xr>.

Calculation of structural models

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Tasks

• Link to Task 01: Canavan Disease
• Link to Task 02: Alignments
• Link to Task 03: Sequence-based Predictions
• Link to Task 04: Structural Alignments
• Link to Task 05: Homology Modelling
• Link to Task 06: Protein Structure Prediction from Evolutionary Sequence Variation
• Link to Task 07: Researching SNPs
• Link to Task 08: Sequence-based Mutation Analysis
• Link to Task 09: Structure-based Mutation Analysis
• Link to Task 10: Normal Mode Analysis
• Link to Task 11: Molecular Dynamics Simulation