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The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. Technical details are reported in our [[Gaucher_Task07_Protocol|protocol]]. == Cystral structure == <figtable id="tab:mutations"> {| style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 2px 0 2px 0; text-align:center" width="400px" |- style="background-color: lightgrey" ! PDB !! Res [Å] !! R value !! Coverage !! pH |- | colspan=5 style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 1px 0 0 0"| |- style="background-color:lightcoral" | [http://www.rcsb.org/pdb/explore/explore.do?structureId=2nt0 2nt0] || 1.80 || 0.18 || 96% (40-536) || 4.5 |- | [http://www.rcsb.org/pdb/explore/explore.do?structureId=3gxi 3gxi] || 1.84 || 0.19 || 96% (40-536) || 5.5 |- | [http://www.rcsb.org/pdb/explore/explore.do?structureId=2v3f 2v3f] || 1.95 || 0.15 || 96% (40-536) || 6.5 |- | [http://www.rcsb.org/pdb/explore/explore.do?structureId=2v3d 2v3d] || 1.96 || 0.16 || 96% (40-536) || 6.5 |- | [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ogs 1ogs] || 2.00 || 0.18 || 96% (40-536) || 4.6 |} <caption>The 5 crystral structures of glycosylceramidase with the highest resolution. The physiological lysosomal pH value is 4.5. 2nt0 was selected for the analysis.</caption> </figtable> == Mutations == <figtable id="tab:mutations"> {| style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 2px 0 2px 0; text-align:center" width="400px" |- style="background-color: lightgrey" ! Nr !! Pos<br/>P04062 !! Pos<br/>2nt0_A !! From !! To !! Disease<br/>causing |- | colspan=6 style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 1px 0 0 0"| |- | 1 || 99 || 60 || H || R || No |- | 2 || 211 || 172 || V || I || No |- | 3 || 150 || 111 || E || K || Yes |- | 4 || 236 || 197 || L || P || Yes |- | 5 || 248 || 209 || W || R || Yes |- | 6 || 509 || 470 || L || P || No |- | 7 || 351 || 312 || W || C || Yes |- | 8 || 423 || 384 || A || D || Yes |- | 9 || 482 || 443 || D || N || No |- | 10 || 83 || 44 || R || S || No |} <caption>Mutations used for the structure-based mutation analysis.</caption> </figtable> <figure id="fig:mutations"> [[File:mutations.png|thumb|left|400px|<caption>2nt0_A with the selected mutations used for the structure-based analysis. Blue: wildtype residues; Red: mutant residues; Orange: active site residues E235 and E340.</caption>]] </figure> <br clear="all"/> == SCWRL == We employed SCWRL <ref name="scwrl">Qiang Wang, Adrian A. Canutescu, and Roland L. Dunbrack, Jr.(2008). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682191/ SCWRL and MolIDE: Computer programs for side-chain conformation prediction and homology modeling]. Nat Protoc.</ref> for substituting the wildtype residues listed in <xr id="tab:mutations"/> by the corresponding mutatant residues which are chosen from a rotamer library. <xr id="fig:scwrl"/> denotes the results. <figure id="fig:scwrl"> <gallery perrow=5 widths="100"> File:scwrl_1.png|1: H60R (2.88) File:scwrl_2.png|2: V172I (4.39) File:scwrl_3.png|3: E111K (8.14) File:scwrl_4.png|4: L197P (3.28) File:scwrl_5.png|5: W209R (9.23) File:scwrl_6.png|6: L470P (28.84) File:scwrl_7.png|7: W312C (0.18) File:scwrl_8.png|8: A384D (13.50) File:scwrl_9.png|9: D443N (2.36) File:scwrl_10.png|10: R44S (4.85) </gallery> <caption>Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; In brackets: energy(mutant)-energy(wildtype).</caption> </figure> None of rotamers chosen by SCWRL clashed with another side-chain or the backbone. The only mutation which led to a structural change was '''L470P'''. Here, the insertion of proline interrupted the beta-sheet. The hydrogen bonding network changed in case of mutation number 1, 5, 7, and 8 (cf. <xr id="tab:scwrl"/>). '''W209R''' introduces a hydrophilic arginine which forms a hydrogen bond to T180. Although not predicted by SCWRL, the arginine might impact the protein structure. '''W312C''' is located next to the active site (cf. <xr id="fig:mutations"/>) and there exists a hydrogen bond to E340. Substitution the hydrophobic tryptohphane by a hydrophlic cysteine in the vicinity of the active site might account for the disease-causing effect of this mutation. As expected, all mutations increased the energy of the model (cf. the energy difference in brackets in <xr id="fig:scwrl"/>). The energy increased most in case of '''L470P''' due to the break of the beta-sheet. '''A384D''' and '''W209R''' also made the model less stable which is caused by substituting an unpolar residue by a charged residue. All four mutations which increased the model energy most are disease-causing. <figtable id="tab:scwrl"> {| style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 2px 0 2px 0; text-align:center" width="700px" |- style="background-color: lightgrey" ! rowspan="2" | Nr !! rowspan="2" style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Mutation !! colspan="2" style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Wildtype !! colspan="2" style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Mutatant !! rowspan="2"| Clashes !! rowspan="2" | Structural<br/>change |- style="background-color: lightgrey" ! H-bonds !! style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobicity !! H-bonds !! style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobicity |- | colspan=8 style="border-collapse: separate; border-style: solid; border-spacing: 0; border-width: 1px 0 0 0"| |- | 1 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | H60R | T471 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic | G62 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 2 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | V172I | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic || No || No |- | 3 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | E111K | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 4 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | L197P | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic || No || No |- | 5 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | W209R | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | T180 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 6 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | L470P | T482 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | T482 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic || No || Yes |- | 7 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | W312C | E340, C342, P316 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | E340, C342 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 8 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | A384D | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophobic | V404 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 9 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | D443N | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic | || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic || No || No |- | 10 | style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | R44S | S13, Y487 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic | S13, Y487 || style="border-style: solid; border-spacing: 0; border-width: 0 1px 0 0;" | Hydrophilic | No || No |} <caption>Structure-based analysis of SNPs from <xr id="tab:mutations"/>. H-bonds: residues involved in forming hydrogen bonds (cut-off: 3.2 Å).</caption> </figtable> We further noticed that SCRWL changed the backbone at some positions which led to different secondary structure assignments (<xr id="fig:scwrl_ss"/>). The positions at which the deviations could be observed were independent from the mutated sites. <figure id="fig:scwrl_ss"> [[File:scwrl_ss.png|thumb|150px|<caption>Seconary structure elements of 2nt0_A (grey) compared to secondary structure elements of models built by SCRWL.</caption>]] </figure> == FoldX == The superposition of the rotamer configurations predicted by FoldX and SCWRL are shown in <xr id="fig:foldx"/>. The predictions of both tools differed in case of four mutations. In case of '''H60R''', the side-chain orientation of arginine predicted by FoldX forms two instead one hydrogen bonds to T741 and might therefore impact the protein structure more than the orientation of SCRWL. In case of '''A384D''', the romater of FoldX might be more stable than the one of SCWRL since it has a higher distance to the surrounding residues. In case of '''D443N''' we prefer the prediction of SCWRL which is closer to the wildtype configuration. For the same reason we prefer the prediction of FoldX in case of '''R442'''. For the subsequent GROMACS analysis, we hence chose the FoldX model in case of mutation number 8 and 10 and the SCWRL models for all all other mutations. <figure id="fig:foldx"> <gallery perrow=5 widths="100"> File:foldx_1.png|1: H60R (-0.39) File:foldx_2.png|2: V172I (-0.80) File:foldx_3.png|3: E111K (-0.36) File:foldx_4.png|4: L197P (0.28) File:foldx_5.png|5: W209R (2.86) File:foldx_6.png|6: L470P (8.59) File:foldx_7.png|7: W312C (1.88) File:foldx_8.png|8: A384D (4.52) File:foldx_9.png|9: D443N (-0.34) File:foldx_10.png|10: R44S (0.60) </gallery> <caption>Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; Orange: rotamer FoldX; In brackets: energy(mutant)-energy(wildtype).</caption> </figure> A comprehensive list of the differences between the mutant and the wildtype models can be found [[FoldX Difference Mutant/Wildtype|here]]. The total energy increased in case of mutation number 4-8, and 10. Just as in case of SCWRL (cf. <xr id="fig:scwrl"/>), '''L470P''', '''A384D''', and '''W209R''' increased the energy of the model most. Since it is unlikely that mutations like '''V172I''' decrease the energy, we consider the energy calculations of SCWRL as more plausible. == Minimise == {| |<figure id="fig:minmise_scwrl_energies">[[File:minimise_scwrl_energies.png|thumb|300px|<caption>Energy of the SCWRL models vs. the number of <tt>minimise</tt> iterations.</caption>]]</figure> |<figure id="fig:minmise_foldx_energies">[[File:minimise_foldx_energies.png|thumb|300px|<caption>Energy of the FoldX models vs. the number of <tt>minimise</tt> iterations.</caption>]]</figure> |} <figure id="fig:minimise_scwrl_mutations"> <gallery perrow=5 widths="100"> File:minimise_scwrl_1.gif|1: H60R File:minimise_scwrl_2.gif|2: V172I File:minimise_scwrl_3.gif|3: E111K File:minimise_scwrl_4.gif|4: L197P File:minimise_scwrl_5.gif|5: W209R File:minimise_scwrl_6.gif|6: L470P File:minimise_scwrl_7.gif|7: W312C File:minimise_scwrl_8.gif|8: A384D File:minimise_scwrl_9.gif|9: D443N File:minimise_scwrl_10.gif|10: R44S </gallery> <caption>Side-chain optimization of SCWRL models over five iterations <tt>minimise</tt>. Green: the input model.</caption> </figure> <figure id="fig:minimise_foldx_mutations"> <gallery perrow=5 widths="100"> File:minimise_foldx_1.gif|1: H60R File:minimise_foldx_2.gif|2: V172I File:minimise_foldx_3.gif|3: E111K File:minimise_foldx_4.gif|4: L197P File:minimise_foldx_5.gif|5: W209R File:minimise_foldx_6.gif|6: L470P File:minimise_foldx_7.gif|7: W312C File:minimise_foldx_8.gif|8: A384D File:minimise_foldx_9.gif|9: D443N File:minimise_foldx_10.gif|10: R44S </gallery> <caption>Side-chain optimization of FoldX models over five iterations <tt>minimise</tt>. Green: the input model.</caption> </figure> == Gromacs == === Runtime analysis === To show the relationship between nsteps and runtime of 'mdrun', different nstep were chosen from 50 to 1000. Three different energy functions were selected: AMBER03 protein, nucleic AMBER94 CHARMM27 all-atom force field (with CMAP) OPLS-AA/L all-atom force field nstep=50 real 0m7.426s user 0m13.230s sys 0m1.020s nstep=100 real 0m14.136s user 0m26.040s sys 0m1.440s nstep=200 real 0m27.229s user 0m51.140s sys 0m2.660s nstep=300 nstep=400 nstep=500 nstep=600 nstep=700 nstep=800 nstep=900 nstep=1000 === Mutations === Mutation 1 Energy Average Err.Est. RMSD Tot-Drift ------------------------------------------------------------------------------- Bond 1624.88 820 5492.83 -5022.62 (kJ/mol) Angle 4289.66 76 402.822 -411.894 (kJ/mol) Potential -45289.6 2900 16896.6 -18817.6 (kJ/mol) == References == <references/>

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