Difference between revisions of "Structure-based mutation analysis (PKU)"

Latest revision as of 11:49, 29 August 2012

To all who are engaged in Psyche's task, of sorting out the seeds of good from the seeds of evil, i dedicate this discourse. FRASER

Short task description

This week, we will introduce mutations in the known tertiary structure of our protein and calculate and compare the potential energy of mutant and wildtype protein with different methods. See the complete task description for details. Our journal might be found here.

Finding the right structure

As proposed we searched the UNIProt entry for our protein and then selected the entry with the highest resolution and the lowest r-Value. In our case this is 1J8U which is the protein in a complex with its cosubstrate BH4. IN the following we will only use this structure, but we also list the results we found. <figtable id="tab:uniprotresult">

Table with results from UNIProt with r-Value inserted

Entry	Method	Resolution (Å)	r-Value	Chain	Positions	PDBsum
1DMW	X-ray	2.00	0.200	A	118-424	[»]
1J8T	X-ray	1.70	0.197	A	103-427	[»]
1J8U	X-ray	1.50	0.157	A	103-427	[»]
1KW0	X-ray	2.50	0.220	A	103-427	[»]
1LRM	X-ray	2.10	0.211	A	103-427	[»]
1MMK	X-ray	2.00	0.199	A	103-427	[»]
1MMT	X-ray	2.00	0.213	A	103-427	[»]
1PAH	X-ray	2.00	0.176	A	117-424	[»]
1TDW	X-ray	2.10	0.206	A	117-424	[»]
1TG2	X-ray	2.20	0.213	A	117-424	[»]
2PAH	X-ray	3.10	0.251	A/B	118-452	[»]
3PAH	X-ray	2.00	0.175	A	117-424	[»]
4ANP	X-ray	2.11	0.204	A	104-427	[»]
4PAH	X-ray	2.00	0.169	A	117-424	[»]
5PAH	X-ray	2.10	0.163	A	117-424	[»]
6PAH	X-ray	2.15	0.171	A	117-424	[»]

</figtable> In <xr id="tab:uniprotresult"/> there are all results according to which we selected 1J8U to be our reference for this weeks task. The corresponding line is marked in yellow. Unfortunately, as we discovered in the preparation of SCWRL, the coverage in not from 103 to 427 for 18JU but only from 118 to 427.

1J8U

In order to know the structure of the protein and its important residues, we have a look at its structure with PyMol and visualize the BH4 and the Fe-ion with the most important residues.

Rendering of the overall structures of 1J8U using PyMol. The protein is colored cyan overall, whereas the Fe-atom is colored red and the important residues are shown as sticks and colored in the element-based fashion. Binding is shown with yellow strokes, if the distance is bigger than 1.5 Å

</figure>

Rendering of a close-up of the structures of 1J8U using PyMol. The protein is colored cyan overall, whereas the Fe-atom is colored red and the important residues are shown as sticks and colored in the element-based fashion. Binding is shown with yellow strokes, if the distance is bigger than 1.5 Å

</figure>

Structure of 1J8U with both ligands Iron (red) and BH4 (element coded color) with their binding sites orange and yellow respectively. BH4-binding sites are van-der-Waal's based and hydrogen bond based, whereas iron is covalently bound (orange)

</figure>

Mutations

As you probably know from last weeks dataset the first two mutations are located before residue 103 and therefore not contained in the structure. We changed them to mutations, which we think are interesting from a structural view. We propose the following dataset, chosen mostly from well known SNPs from OMIM. They include mutations causing no reported effect, the mild related hyperphenylalaninemia (reduced activity, but functional enzyme) and phenylketonuria. We will present a similar table for each method, in which we show the results we would suggest from this method alone in the end we present our overall results here, which will be a intuitive consensus for all the intermediate results.

SNP	effect	prediction	validation
ARG158GLN	disease causing	effect
GLN172HIS	non-disease	no effect
ARG243GLN	disease causing	effect
LEU255SER	disease causing	effect
MET276VAL	non-disease	effect
ARG297CYS	disease causing	no effect
ALA322GLY	hyperphenylalaninemia	no effect
GLU330ASP	disease causing	effect
GLY337VAL	disease causing	effect
ARG408TRP	disease causing	effect

ARG158GLN

The bindingsites are colored according to the overview picture above the mutation (ARG158GLN)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>This mutation, which is diseasecausing is in a moderate distance to the important binding sites (<xr id="fig:ARG158GLNmutation" />). A clear explanation, why this has an effect on the protein can not be found from the structure alone. But even with the results from last weeks Task, we just had to rely on the clashes which occurred when we estimated the structure.

GLN172HIS

The bindingsites are colored according to the overview picture above the mutation (GLN172HIS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>In <xr id="fig:ARG158GLNmutation" /> one can see the big distance between the binding sites and the mutation location. Since we know that this is harmless, one would tend to say, that its clear, due to the distance, but as we know, that we also have mutations in a big distance which cause the disease, we are bound to say we do not know the mechanism.

ARG243GLN

The binding sites are colored according to the overview picture above the mutation (ARG243GLN) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>This is the second mutation of this kind, which means a change from arginine to glycine. As the other mutation, this one is disease causing. But in difference to the other case, this one is rather close to the binding areas (see <xr id="fig:ARG243GLNmutation" /> )

LEU255SER

The binding sites are colored according to the overview picture above the mutation (LEU255SER)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>When one compares this mutation ( in <xr id="fig:LEU255SERmutation" />) to the reference structure in <xr id="fig:structuremoving" /> one can see, that one of the yellow residues (BH4 binding site) is mutated. And as one would expect this mutation is harmful.

MET276VAL

The binding sites are colored according to the overview picture above the mutation (MET276VAL) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the mutation shown in <xr id="fig:MET276VALmutation" /> is rather far from any important binding site, one would expect it to be of little effect for the protein, which is the case for this mutation.

ARG297CYS

The binding sites are colored according to the overview picture above the mutation (ARG297CYS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the last mutation, this one is located quite far from any annotated sites ( <xr id="fig:ARG297CYSmutation" />), however it is located in the kink of two helices, and therefore the expectation which this mutation might have on the protein tends towards disease causing, which is actually the right choice.

ALA322GLY

The binding sites are colored according to the overview picture above the mutation (ALA322GLY) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure> The mutation, which affects one of the amino acids responsible for the BH4-binding, is shown in <xr id="fig:ALA322GLYmutation" /> and as one would guess is disease causing.

GLU330ASP

The binding sites are colored according to the overview picture above the mutation (GLU330ASP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>As the last mutation in <xr id="fig:ALA322GLYmutation" /> this mutation affects a binding site (this time iron) and is disease causing.

GLY337VAL

The binding sites are colored according to the overview picture above the mutation (GLY337VAL) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>This mutation (shown in <xr id="fig:GLY337VALmutation" />) is far from any known binding site and located in a loop on the far outside of the protein, but it is disease causing.

ARG408TRP

The binding sites are colored according to the overview picture above the mutation (ARG408TRP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.

</figure>In <xr id="fig:ARG408TRPmutation" /> one can again see a mutation which is pretty far from the annotated sites, but still is disease causing.

SCWRL

preparations

We use the repairPDB script to extract the sequence from our model file. Afterwards we do some bashing to get the sequence in small letters but for the mutation, which has to be in capitals. <source lang="bash"> $ repairPDB 1J8U.pdb -seq > 1J8U.seq $ tr '[:upper:]' '[:lower:]' < 1J8U.seq > lower18JU.seq $ sed 's/^$.\{<pos-118>\}$<original in small letter>$.*$/\1<target in capital letter>\2/' lower18JU.seq > lower18JUmut<pos>.seq </source>

usage

Afterwards, we used a little bashscript which just applies all SCWRL-calls after another. In the following you see one example: <source lang="bash"> $ /opt/SS12-Practical/scwrl4/Scwrl4 -i ../1J8U.pdb -s lower18JUmut158.seq -o mut158.pdb > mut158.log </source>

results

The following pictures show the structure optimized by SCWRL. The mutant is colored in red for this residue as the wildtype is colored in green, as the structure apart from this residue is unchanged, the color is chosen automatically by PyMol. Polar bonds are colored respectively to mutant and wildtype, but note, that there might be overlaps, where only one color is shown.

Mutation ARG158GLN: one can see, that with the mutation bonds that were formed before can no longer be formed and new ones are established
Mutation GLN172HIS: as in Mutation ARG158GLN the amount of bonds is altered
Mutation ARG243GLN: as well as in all the other results up to now, the mutant can form one bond less than the wildtype
Mutation LEU255SER: in this picture one can see, that the mutant can form more bonds than the wildtype did
Mutation MET276VAL: as before the mutation results in one bond more.
Mutation ARG297CYS: here one can not see any change apart from the amino acids
Mutation ALA322GLY: as the Glycine is completely contained in the Alanine, no change can be noticed
Mutation GLU330ASP: due to the change of amino acids the amount of bonds decreases by two
Mutation GLY337VAL: As the mutant completely contains the wildtype Glycine no changes can be noticed
Mutation ARG408TRP: With the change of amino acids all bonds which were formed before can not be established anymore

Comparison of all the energies for the mutations and wildtype for scwrl

Mutation	energy	energy mut/energy wt	prediction
wildtype	164.21	1	no effect
ARG158GLN	172.845	1.05	effect
GLN172HIS	169.699	1.03	no effect
ARG243GLN	152.987	0.93	effect
LEU255SER	166.123	1.01	effect
MET276VAL	164.472	1.00	effect
ARG297CYS	166.646	1.01	no effect
ALA322GLY	164.285	1.00	no effect
GLU330ASP	171.68	1.04	effect
GLY337VAL	164.304	1.00	no effect
ARG408TRP	393.804	2.39	effect

</figtable>

foldX

The other approach we used is foldX

preparation

We use the run.txt from the example at the test and adapt the list and individual list as its shown in the journal.

usage

scwrl -runfile run.txt

</source>

results

As we already have results from the SCWRL run, we just compare those with the new results from foldX. The comparison with the sourounding structures is left for the reader as an exercise. The residue placed by SCWRL is colored in blue whereas the foldX residue is orange. THe surrounding structure is colored randomly by PyMol. <figure id="foldxstructcompare">

In this Figure, we show the differences in the structures which we got from both methods, where scwrl is colored in blue and foldX is colored in cyan. This is the only change and we expect it to have a very low effect on the activity of the protein, because no residues which are known for their importance are found in this region

</figure>

Mutation ARG158GLN: In this picture the changes between foldX and SCWRL are not big. The stuctural differences of the two residues result in the fact, that SCWRl includes H-atoms which are not included in foldX. Apart from that, the residues are twisted which results in a difference of the H-bonds. As one can see, SCWRL forms a bond (colored in blue) which is not present in the residue conformation in foldX.
Mutation GLN172HIS: Here the changes again are only a small change in the angle of the residue and the "missing" H-atom in the foldX-structure (orange). No other changes are obvious.
Mutation ARG243GLN: Due to the change in the conformation of the residues, the orange structure (foldX) can form a bond, whereas the blue structure is not able to form any polar bonds.
Mutation LEU255SER: In this case, in spite of the completely changed structures, both of the conformations are able to establish bonds to the same target, But as they are formed from a different angle, we expect this to change the structure through torsion of the molecules.
Mutation MET276VAL: As the structure derived from SCWRL (blue) can form the blue bond, which can not be formed in the structure from foldX (orange), the structure of SCWRL is probably quite different from the other one.
Mutation ARG297CYS: With the torsion of the angle of the two residues, they seem to have a quite different properties, but the changes seem to be rather minor if one has a look at the surroundings.
Mutation ALA322GLY: In this case there is no difference in the structures derived from both methods
Mutation GLU330ASP: Similar to the case ARG158GLU the structure which is proposed by SCWRL can build one more polar bond, than the corresponding structure.
Mutation GLY337VAL: Again the changes seem to be rather big, but there are no visible changes in the bonds or clashes
Mutation ARG408TRP: This is the only mutation, in which the two methods support completly different structures, which do not yield any clashes or bonds, which are possible in the one and not in the other.

Comparison of all the energies for the mutations and wildtype for foldX

Mutation	energy	energy mut/energy wt	energy mut/energy relative wt	prediction
wildtype	14.00	1	1	no effect
ARG158GLN	6.36	0.45	1.31	effect
GLN172HIS	16.69	1.16	0.99	no effect
ARG243GLN	6.15	0.43	0.72	effect
LEU255SER	23.57	1.68	1.21	effect
MET276VAL	21.99	1.57	1.06	effect
ARG297CYS	20.31	1.45	1.01	no effect
ALA322GLY	21.76	1.55	1.05	no effect
GLU330ASP	19.65	1.40	1.02	effect
GLY337VAL	24.01	1.71	1.10	effect
ARG408TRP	34.36	2.45	1.58	effect

</figtable>

minimise

preparation

To use minimise we have to clear the structure of hydrogen atoms and solvent which we do again using the repairPDB script. <source lang="bash"> $ repairPDB 1J8U.pdb -noh -nosol > 1J8U_pure.pdb </source>

usage

Afterwards a little script is used to apply the minimization five times and each time using the output as input.

usage:

<source lang="bash"> $ runAllMinimiser.sh <filestem> </source>

script

results

Just to see the differences between the results of one run and several using the output again as input, we produce this little animated .gif, which is basicly all the results from the 18JU.pdb on till the fifth minimise result. <figure id="fig:helixminimise">

Superimposition of all structures derived from minimise for 1J8U which means the wildtype. For a better comparison first the wildtype is shown in blue. Then it will get transparent and the first result of minimize is shown in teal. Then the transparent wildtype disappears and the first result will get transparent and the second result will show. This continues until the final result which is red. Then the animation will start again.

</figure> In <xr id="fig:helixminimise" /> one can see that minimise shifts the start of the helix further down in the picture, and once it reaches a certain point, the start of the helix is converted to a coiled region instead. A similar effect can be found if one compares sheets in the output of the different minimise runs. Here a twisting of the sheets takes place, which can bee seen in <xr id="fig:sheetminimise" /> <figure id="fig:sheetminimise">

Structure of 1J8U before (cyan) and after (red) minimise. The red structure is the result of five consecutive runs of minmise, and shows a twist in the orientation of the sheet.

</figure> <figure id="fig:timecourseminimise">

This picture shows the time course of the energy derived from minimise for 14 steps for the 1J8U-structure we got from the pdb and excluded the solvent

</figure> As one can see in <xr id="fig:timecourseminimise" /> consecutive multiple minimise runs do not decrease the energy but increase it drastically. But the first two minimise steps reduce the energy, and therefore we want to compare the energyoutput from minimise to the two others found in <xr id="tab:foldxenergies" /> and <xr id="tab:scwrlenergies" />. <figtable id="tab:minimiseenergies">

Comparison of all mutations with energies derived from minimise

mutation	energy foldX	energy foldX/energy wt	energy scwwrl	energy scwrl/energy wt	prediction
wildtype	-7516.267118				no effect
ARG158GLN	-7564.267932	1.00	-7400.825142	0.98
GLN172HIS	-7513.718344	0.99	-7375.087666	0.98
ARG243GLN	-7522.768865	1.00	-7476.255952	0.99
LEU255SER	-7522.768865	1.00	-7476.255952	0.99
MET276VAL	-7502.369873	0.99	-7374.091542	0.98
ARG297CYS	-7616.286033	1.01	-7484.442763	0.99
ALA322GLY	-7509.307761	0.99	-7389.927555	0.98
GLU330ASP	-7494.936652	0.99	-7367.788205	0.98
GLY337VAL	-7504.533976	0.99	-7380.216180	0.98
ARG408TRP	-5289.743707	0.70	-5438.301688	0.72	effect

</figtable> TODO: this and maybe compare with the other weird energy outputs from SCWRL and foldX und dann halt irgendwie daraus die prediction für die einzelnen Mutationen ableiten

surface considerations

As we only can get very little information from the minimise-results, we try to include the overall surface of the protein as well as the important sites into the prediction of the effectivity. In the following, mutants are colored red and wildtype residues green. The wildtype surface is shown in green and grey (for non mutant areas). In contrast the mutant surface will be shown as a mesh and in red color.

Mutation ARG158GLN: As the arginine is a rather long residue, which sticks out of the protein in most cases, here we can find some kind of small hill arising in green, where the red mesh stays rather flat due to the dens structure of glutamine
Mutation GLN172HIS: As the plain structures of the mutant in red and the wildtype residue in green are quite different the result in surface is not big, the only thing which is changing, is that the green bulge is now in a silightly different angle.
Mutation ARG243GLN: In this picture we see the big differences which come with the change of the amino acids. As the normal surface is kind of a small tunnel on the upper left, with the change to glutamine this tunnel would widen (see red mesh) and a new hole on the right would appear, which is not present in the green surface (wildtype)
Mutation LEU255SER: Like in the picture before (ARG243GLN) with the mutation comes a bigger hole in the structure, which is due to the smaller size of the new amino acid
Mutation MET276VAL: This mutation yields big changes in the structure, as the wildtype (green) formed a slightly descending hyperplane, the red mesh shows a bulge outof the structure which is rising where the wildtype was falling.
Mutation ARG297CYS: With the rather long arginine substituted with a in comparison small cysteine, the apex which was formed by arginine disappears and instead forms a small dent
Mutation ALA322GLY: Again due to the change of a bigger residue to a smaller one, the hole in the protein gets widened
Mutation GLU330ASP: Like in ALA322GLY the mutant residue (red) widens the space in the protein
Mutation GLY337VAL: As the mutant residue is bulkier than the original one, the peak which was present in the wildtype (green) also get bulkier
Mutation ARG408TRP: although the amino acid length between arginine and tryptophan is pretty much the same, their width is different. Due to this fact, a new rift in the protein opens and the overall conformation of this part changes, as one can see with the large mesh around the red mutant residue

Comparison of the surface changes with respect to known binding sites and location

Mutation	surface changes	bindingsite	prediction
ARG158GLN	mild to big	no	effect
GLN172HIS	mild	no	no effect
ARG243GLN	big	no	effect
LEU255SER	mild	yes	effect
MET276VAL	big	no	no effect
ARG297CYS	big	no	effect
ALA322GLY	mild	yes	effect
GLU330ASP	mild	yes	effect
GLY337VAL	big	no	effect
ARG408TRP	huge	no	effect

</figtable>

Gromacs

We choose the scwrl structures as input, since there is little difference to observe so far, but scwrl will be used in next week's task and results will be more comparable that way. The provided fetchPDB-script just wgets the gzipped PDBfile for a given PDB identifier, but was not used, since we already had our input structures. To extract the protein alone from the PDB-file, we used repairPDB with the -jprot option. repairPDB provides a number of usefull options, e.g. it extracts or removes DNA, crystal water, selects individual chains or renumbers the sequence.

The minimization preparation and runs were performed with this script. There is also a short script to run g_energy from the .edr-files here. We initially choose the forcefields TIP3P for water, AMBER03 (option 1 in the interactive menu), CHARMM27 (option 8) and GROMOS96 53a6 (option 13) for the protein, since they are amongst the most popular, but run into troubles with GROMOS and replaced it with OPLS-AA (option 14). All of them work with our chosen water model TIP3P. We used the provided standard configuration file for gromacs, described elsewhere.

Results

Runtime

For most mutants and most forcefields the minimization converges between step 250 and 300 and calculation stops then, so there is little sense in starting with a value of more then say 1000 for nsteps. If the energy does not converge by then, we'd suspect some error in the input. We give a few runtimes for the wildtype and the AMBER03 forcefield in <xr id="fig:runtime"/>, performed on the lrz grid. The maximum runtime we observed was around 50 seconds (not shown), the average runtime for a minimization is around 30 seconds. Since the calculation of every step requires the same amount of work, the expected runtime behaviour until convergence is perfectly linear in nsteps plus a constant for initialization. The provided datapoints confirm this nicely. <figure id="fig:runtime">

Runtime for different values for nsteps of minimization with Gromacs, using the AMBER03 forcefield and our wildtype 1J8U.

</figure>

Energies

We show the curve of the angle, bond and potential energy during the wildtype minimization for all three forcefields in <xr id="fig:1J8U_energies"/>. In all cases, the energy goes down steeply at first. While the bond energy does not change much after that and potential energy slowly converges, the angle energy rises during optimization until it also converges at a level slightly higher than its initial lowest point. This is as expected, since the total energy is optimized by adjusting the conformation of the protein. The mutants behave qualitatively similar, plots will be available here, once they are uploaded.

The tables <xr id="tab:gromacs_energies_1J8U"/> to <xr id="tab:gromacs_energies_mut408"/> show the energies of angle, bond and the potential energy of the wildtype and the ten mutants. In brackets, the difference to the wildtype is given. The bond energy comprises a small part of the total energy and changes not much in the mutants (the largest change in ARG158GLN is 2.6%). Neither direction nor amplitude of the energy changes seem to correlate with malignance of the mutations directly.

For the AMBER force field the average change in potential energy for the mutants is +304 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +139 kJ/mol. On the other hand, the smallest change in energy is observed in a disease causing, the GLY337VAL mutation. All but one disease causing mutation increase the potential energy, but two of the three benign ones do the same.

So far, we observe that smaller changes in energy indicate a less severe impact on the structure, but the prediction value appears limited, since the false positives number (benign mutations thought to be malign) is high. Setting a cut-off of the potential energy change to 100 kJ/mol would correctly classify five disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 70%.

For the CHARMM force field the average change in potential energy for the mutants is +712 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +101 kJ/mol. The largest change in a benign mutation is only +160.53 kJ/mol and there are four extreme changes above 900 kJ/mol in the malign mutations. Setting a cut-off of the potential energy change to 100 kJ/mol would correctly classify six disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 80%.

For the OPLS-AA force field the average change in potential energy for the mutants is +151 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +57 kJ/mol. The largest change in a benign mutation is +147.33 kJ/mol and there are three larger changes above 100 kJ/mol in the malign mutations, but also one change of +147 kJ/mol in an benign mutation. Setting a cut-off of the potential energy change to 50 kJ/mol would correctly classify five disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 70%.

In all force fields, the largest change is caused by the malign ARG408TRP mutation, a visibly extreme disruption of the structure. Rather surprising is the large change in the benign GLN172HIS, that is located in a coiled region at the outside of PheOh and was one of the easier cases in last week's prediction task.

<figtable id="fig:1J8U_energies"> Course of the energies during minimization of 1J8U with gromacs.

Energie of the angles in 1J8U in the AMBER03 forcefield.	Energie of the bonds in 1J8U in the AMBER03 forcefield.	Potential energy in 1J8U in the AMBER03 forcefield.
	Energie of the bonds in 1J8U in the CHARMM27 forcefield.	Potential energy in 1J8U in the CHARMM27 forcefield.
Energie of the angles in 1J8U in the OPLS-AA forcefield.	Energie of the bonds in 1J8U in the OPLS-AA forcefield.	Potential energy in 1J8U in the OPLS-AA forcefield.

</figtable>

<figtable id="tab:gromacs_energies_1J8U"> Energies after Gromacs minimization of the wildtype.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2403.47 (Δ 0.00)		2092.75 (Δ 0.00)
Bonds (kJ/mol)	522.31 (Δ 0.00)	1162.70(Δ 0.00)	545.17 (Δ 0.00)
Potential (kJ/mol)	-37536.96 (Δ 0.00)	-40392.46(Δ 0.00)	-65323.58 (Δ 0.00)

</figtable>

<figtable id="tab:gromacs_energies_mut158"> Energies after Gromacs minimization of the disease causing ARG158GLN mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2387.31 (Δ -16.17)		2087.86 (Δ -4.90)
Bonds (kJ/mol)	509.25 (Δ -13.06)	1138.40(Δ -24.30)	543.58 (Δ -1.59)
Potential (kJ/mol)	-37171.73 (Δ 365.23)	-39342.72(Δ 1049.75)	-65223.57 (Δ 100.01)

</figtable>

<figtable id="tab:gromacs_energies_mut172"> Energies after Gromacs minimization of the benign GLN172HIS mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2479.53 (Δ 76.06)		2106.61 (Δ 13.86)
Bonds (kJ/mol)	522.68 (Δ 0.37)	1154.75(Δ -7.95)	546.84 (Δ 1.67)
Potential (kJ/mol)	-37163.15 (Δ 373.82)	-40231.94(Δ 160.53)	-65176.25 (Δ 147.33)

</figtable>

<figtable id="tab:gromacs_energies_mut243"> Energies after Gromacs minimization of the disease causing ARG243GLN mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2398.46 (Δ -5.01)		2086.91 (Δ -5.84)
Bonds (kJ/mol)	517.89 (Δ -4.43)	1144.20(Δ -18.50)	542.42 (Δ -2.76)
Potential (kJ/mol)	-37333.11 (Δ 203.86)	-39440.42(Δ 952.05)	-65231.15 (Δ 92.43)

</figtable>

<figtable id="tab:gromacs_energies_mut255"> Energies after Gromacs minimization of the disease causing LEU255SER mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2415.15 (Δ 11.68)		2092.62 (Δ -0.13)
Bonds (kJ/mol)	530.22 (Δ 7.91)	1150.95(Δ -11.75)	546.59 (Δ 1.41)
Potential (kJ/mol)	-37613.71 (Δ -76.75)	-40227.79(Δ 164.67)	-65323.13 (Δ 0.45)

</figtable>

<figtable id="tab:gromacs_energies_mut276"> Energies after Gromacs minimization of the benign MET276VAL mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2402.78 (Δ -0.70)		2094.12 (Δ 1.37)
Bonds (kJ/mol)	522.62 (Δ 0.31)	1153.45(Δ -9.25)	548.90 (Δ 3.72)
Potential (kJ/mol)	-37460.79 (Δ 76.18)	-40308.56(Δ 83.90)	-65297.64 (Δ 25.95)

</figtable>

<figtable id="tab:gromacs_energies_mut297"> Energies after Gromacs minimization of the disease causing ARG297CYS mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2404.12 (Δ 0.65)		2088.02 (Δ -4.73)
Bonds (kJ/mol)	521.46 (Δ -0.86)	1140.64(Δ -22.06)	547.49 (Δ 2.32)
Potential (kJ/mol)	-36943.84 (Δ 593.13)	-39121.65(Δ 1270.81)	-65074.67 (Δ 248.91)

</figtable>

<figtable id="tab:gromacs_energies_mut322"> Energies after Gromacs minimization of the hyperphenylalaninemia causing ALA322GLY mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2409.61 (Δ 6.13)		2091.98 (Δ -0.77)
Bonds (kJ/mol)	528.05 (Δ 5.74)	1147.60(Δ -15.10)	547.21 (Δ 2.03)
Potential (kJ/mol)	-37567.33 (Δ -30.36)	-40334.32(Δ 58.14)	-65325.79 (Δ -2.21)

</figtable>

<figtable id="tab:gromacs_energies_mut330"> Energies after Gromacs minimization of the disease causing GLU330ASP mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2408.24 (Δ 4.77)		2097.44 (Δ 4.69)
Bonds (kJ/mol)	522.71 (Δ 0.39)	1162.17(Δ -0.53)	544.42 (Δ -0.75)
Potential (kJ/mol)	-37373.77 (Δ 163.19)	-40487.35(Δ -94.89)	-65280.02 (Δ 43.57)

</figtable>

<figtable id="tab:gromacs_energies_mut337"> Energies after Gromacs minimization of the disease causing GLY337VAL mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2415.06 (Δ 11.58)		2105.72 (Δ 12.97)
Bonds (kJ/mol)	526.54 (Δ 4.23)	1156.77(Δ -5.93)	548.96 (Δ 3.79)
Potential (kJ/mol)	-37518.37 (Δ 18.60)	-40272.05(Δ 120.41)	-65273.33 (Δ 50.25)

</figtable>

<figtable id="tab:gromacs_energies_mut408"> Energies after Gromacs minimization of the disease causing ARG408TRP mutation.

Type	Amber03	Charmm27	Oplsaa
Angle (kJ/mol)	2430.32 (Δ 26.85)		2110.42 (Δ 17.67)
Bonds (kJ/mol)	524.16 (Δ 1.84)	1150.43(Δ -12.26)	546.33 (Δ 1.15)
Potential (kJ/mol)	-36674.55 (Δ 862.41)	-38873.69(Δ 1518.78)	-64802.38 (Δ 521.21)

</figtable>

@@ Line 1: / Line 1: @@
+''To all who are engaged in Psyche's task, of sorting out the seeds of good from the seeds of evil, i dedicate this discourse.'' '''FRASER'''
 == Short task description ==
-This week, we will introduce mutations in the known tertiary structure of our protein and calculate and compare the potential energy of mutant and wildtype protein with different methods. See the complete [[Task_7_-_Structure-based_mutation_analysis|task description]] for details. Our journal might be found here at some time.
+This week, we will introduce mutations in the known tertiary structure of our protein and calculate and compare the potential energy of mutant and wildtype protein with different methods. See the complete [[Task_7_-_Structure-based_mutation_analysis|task description]] for details. Our journal might be found [[Journal structure based mutation analysis (PKU)|here]].
 == Finding the right structure ==
@@ Line 147: / Line 148: @@
 |}
 </figtable>
-In <xr id="tab:uniprotresult"/> there are all results according to which we selected 1J8U to be our reference for this weeks task. The corresponding line is marked in yellow.
+In <xr id="tab:uniprotresult"/> there are all results according to which we selected 1J8U to be our reference for this weeks task. The corresponding line is marked in yellow. Unfortunately, as we discovered in the [[#preparation|preparation]] of SCWRL, the coverage in not from 103 to 427 for 18JU but only from 118 to 427.
 == 1J8U ==
@@ Line 163: / Line 164: @@
 == Mutations ==
 As you probably know from [[Predicting_the_Effect_of_SNPs_(PKU)#Our dataset|last weeks dataset]] the first two mutations are located before residue 103 and therefore not contained in the structure. We changed them to mutations, which we think are interesting from a structural view.
-We propose the following dataset, chosen mostly from well known SNPs from OMIM. They include mutations causing no reported effect, the mild related hyperphenylalaninemia (reduced activity, but functional enzyme) and phenylketonuria.
+We propose the following dataset, chosen mostly from well known SNPs from OMIM. They include mutations causing no reported effect, the mild related hyperphenylalaninemia (reduced activity, but functional enzyme) and phenylketonuria. We will present a similar table for each method, in which we show the results we would suggest from this method '''alone''' in the end we present our overall results here, which will be a intuitive consensus for all the intermediate results.
 <div style="float: center; width: 100%">
 {| align="center"
@@ Line 172: / Line 173: @@
 |
 |-
-|[[#ARG158GLN|ARG158GLN]]
+| [[#ARG158GLN|ARG158GLN]]
 | disease causing
+| effect
-|
-|[[File:unknown.jpeg|25px|center]]
+| [[File:true.jpeg|25px|center]]
 |-
-|[[#GLN172HIS|GLN172HIS]]
+| [[#GLN172HIS|GLN172HIS]]
 | non-disease
+| no effect
-|
-|[[File:unknown.jpeg|25px|center]]
+| [[File:true.jpeg|25px|center]]
 |-
 |[[#ARG243GLN|ARG243GLN]]
 | disease causing
-|
+| effect
-|[[File:unknown.jpeg|25px|center]]
+| [[File:true.jpeg|25px|center]]
 |-
-|[[#LEU255SER|LEU255SER]]
+| [[#LEU255SER|LEU255SER]]
 | disease causing
-|
+| effect
-|[[File:unknown.jpeg|25px|center]]
+| [[File:true.jpeg|25px|center]]
 |-
 |[[#MET276VAL|MET276VAL]]
 | non-disease
-|
+| effect
-|[[File:unknown.jpeg|25px|center]]
+|[[File:true.jpeg|25px|center]]
 |-
 |[[#ARG297CYS|ARG297CYS]]
 | disease causing
+| no effect
-|
-|[[File:unknown.jpeg|25px|center]]
+|[[File:wrong.jpeg|25px|center]]
 |[[File:new.jpeg|25px|center]]
 |-
 |[[#ALA322GLY|ALA322GLY]]
 | hyperphenylalaninemia
+| no effect
-|
-|[[File:unknown.jpeg|25px|center]]
+|[[File:wrong.jpeg|25px|center]]
 |-
 |[[#GLU330ASP|GLU330ASP]]
 | disease causing
+| effect
-|
-|[[File:unknown.jpeg|25px|center]]
+|[[File:true.jpeg|25px|center]]
 |[[File:new.jpeg|25px|center]]
 |-
 |[[#GLY337VAL|GLY337VAL]]
 | disease causing
-|
+| effect
-|[[File:unknown.jpeg|25px|center]]
+|[[File:true.jpeg|25px|center]]
 |-
 |[[#ARG408TRP|ARG408TRP]]
 | disease causing
-|
+| effect
-|[[File:unknown.jpeg|25px|center]]
+|[[File:true.jpeg|25px|center]]
 |}
@@ Line 229: / Line 229: @@
 === ARG158GLN ===
-This mutation, which is diseasecausing is in a moderate distance to the important binding sites (<xr id="fig:ARG158GLNmutation" />). A clear explanation, why this has an effect on the protein can not be found from the structure alone.
 <figure id="fig:ARG158GLNmutation">
-[[File:ARG158GLNmutation.png|thumb|400px|center|<caption> The bindingsites are colored according to the overview picture above the mutation is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+[[File:ARG158GLNmutation.png|thumb|300px|<caption> The bindingsites are colored according to the overview picture above the mutation (ARG158GLN)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>This mutation, which is diseasecausing is in a moderate distance to the important binding sites (<xr id="fig:ARG158GLNmutation" />). A clear explanation, why this has an effect on the protein can not be found from the structure alone.
-</figure>
+But even with the [[Predicting_the_Effect_of_SNPs_(PKU)#ARG158GLN|results from last weeks Task]], we just had to rely on the clashes which occurred when we estimated the structure.
 === GLN172HIS ===
+<figure id="fig:ARG158GLNmutation">
+[[File:GLN172HISmutation.png|thumb|300px|left|<caption> The bindingsites are colored according to the overview picture above the mutation (GLN172HIS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>In <xr id="fig:ARG158GLNmutation" /> one can see the big distance between the binding sites and the mutation location. Since we know that this is harmless, one would tend to say, that its clear, due to the distance, but as we know, that we also have mutations in a big distance which cause the disease, we are bound to say we do not know the mechanism.
 === ARG243GLN ===
+<figure id="fig:ARG243GLNmutation">
+[[File:ARG243GLNmutation.png|thumb|300px|<caption> The binding sites are colored according to the overview picture above the mutation (ARG243GLN) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>This is the second mutation of this kind, which means a change from arginine to glycine. As the other mutation, this one is disease causing. But in difference to the other case, this one is rather close to the binding areas (see <xr id="fig:ARG243GLNmutation" /> )
 === LEU255SER ===
+<figure id="fig:LEU255SERmutation">
+[[File:LEU255SERmutation.png|thumb|left|300px|<caption> The binding sites are colored according to the overview picture above the mutation (LEU255SER)is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>When one compares this mutation ( in <xr id="fig:LEU255SERmutation" />) to the reference structure in <xr id="fig:structuremoving" /> one can see, that one of the yellow residues (BH4 binding site) is mutated. And as one would expect this mutation is harmful.
 === MET276VAL ===
+<figure id="fig:MET276VALmutation">
+[[File:MET276VALmutation.png|thumb|300px|<caption> The binding sites are colored according to the overview picture above the mutation (MET276VAL) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>As the mutation shown in <xr id="fig:MET276VALmutation" /> is rather far from any important binding site, one would expect it to be of little effect for the protein, which is the case for this mutation.
 === ARG297CYS ===
+<figure id="fig:ARG297CYSmutation">
+[[File:ARG297CYSmutation.png|thumb|300px|left|<caption> The binding sites are colored according to the overview picture above the mutation (ARG297CYS) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>As the last mutation, this one is located quite far from any annotated sites ( <xr id="fig:ARG297CYSmutation" />), however it is located in the kink of two helices, and therefore the expectation which this mutation might have on the protein tends towards disease causing, which is actually the right choice.
 === ALA322GLY ===
+<figure id="fig:ALA322GLYmutation">
-=== GLU330AS P===
+[[File:ALA322GLYmutation.png|thumb|300px|<caption> The binding sites are colored according to the overview picture above the mutation (ALA322GLY) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure> The mutation, which affects one of the amino acids responsible for the BH4-binding, is shown in <xr id="fig:ALA322GLYmutation" /> and as one would guess is disease causing.
+=== GLU330ASP ===
+<figure id="fig:GLU330ASPmutation">
+[[File:GLU330ASPmutation.png|thumb|300px|left|<caption> The binding sites are colored according to the overview picture above the mutation (GLU330ASP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>As the last mutation in <xr id="fig:ALA322GLYmutation" /> this mutation affects a binding site (this time iron) and is disease causing.
+=== GLY337VAL ===
+<figure id="fig:GLY337VALmutation">
+[[File:GLY337VALmutation.png|thumb|left|300px|<caption> The binding sites are colored according to the overview picture above the mutation (GLY337VAL) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>This mutation (shown in <xr id="fig:GLY337VALmutation" />) is far from any known binding site and located in a loop on the far outside of the protein, but it is disease causing.
 === ARG408TRP ===
+<figure id="fig:ARG408TRPmutation">
+[[File:ARG408TRPmutation.png|thumb|300px|<caption> The binding sites are colored according to the overview picture above the mutation (ARG408TRP) is colored in red, and the sticks for this residue as well as for the binding residues are shown as well.</caption>]]
+</figure>In <xr id="fig:ARG408TRPmutation" /> one can again see a mutation which is pretty far from the annotated sites, but still is disease causing.
+<br style="clear:both;">
+== SCWRL ==
+=== preparations ===
+We use the [[repairPDB]] script to extract the sequence from our model file. Afterwards we do some bashing to get the sequence in small letters but for the mutation, which has to be in capitals.
+<source lang="bash">
+$ repairPDB 1J8U.pdb -seq > 1J8U.seq
+$ tr '[:upper:]' '[:lower:]' < 1J8U.seq > lower18JU.seq
+$ sed 's/^\(.\{<pos-118>\}\)<original in small letter>\(.*\)/\1<target in capital letter>\2/' lower18JU.seq > lower18JUmut<pos>.seq
+</source>
+=== usage ===
+Afterwards, we used a little [[Journal structure based mutation analysis (PKU)#scwrl|bashscript]] which just applies all SCWRL-calls after another. In the following you see one example:
+<source lang="bash">
+$ /opt/SS12-Practical/scwrl4/Scwrl4 -i ../1J8U.pdb  -s lower18JUmut158.seq -o mut158.pdb > mut158.log
+</source>
+=== results ===
+The following pictures show the structure optimized by SCWRL. The mutant is colored in red for this residue as the wildtype is colored in green, as the structure apart from this residue is unchanged, the color is chosen automatically by PyMol. Polar bonds are colored respectively to mutant and wildtype, but note, that there might be overlaps, where only one color is shown.
+<center>
+<gallery widths=450px heights=250px perrow=2>
+File:pkuscwrlmutation158.png|Mutation ARG158GLN: one can see, that with the mutation bonds that were formed before can no longer be formed and new ones are established
+File:pkuscwrlmutation172.png|Mutation GLN172HIS: as in Mutation ARG158GLN the amount of bonds is altered
+File:pkuscwrlmutation243.png|Mutation ARG243GLN: as well as in all the other results up to now, the mutant can form one bond less than the wildtype
+File:pkuscwrlmutation255.png|Mutation LEU255SER: in this picture one can see, that the mutant can form more bonds than the wildtype did
+File:pkuscwrlmutation276.png|Mutation MET276VAL: as before the mutation results in one bond more.
+File:pkuscwrlmutation297.png|Mutation ARG297CYS: here one can not see any change apart from the amino acids
+File:pkuscwrlmutation322.png|Mutation ALA322GLY: as the Glycine is completely contained in the Alanine, no change can be noticed
+File:pkuscwrlmutation330.png|Mutation GLU330ASP: due to the change of amino acids the amount of bonds decreases by two
+File:pkuscwrlmutation337.png|Mutation GLY337VAL: As the mutant completely contains the wildtype Glycine no changes can be noticed
+File:pkuscwrlmutation408.png|Mutation ARG408TRP: With the change of amino acids all bonds which were formed before can not be established anymore
+</gallery>
+</center>
+<figtable id="tab:scwrlenergies">
+<center><caption>Comparison of all the energies for the mutations and wildtype for scwrl </caption></center>
+<div style="float: center; width: 100%">
+{| align="center"
+!| Mutation
+!| energy
+!| energy mut/energy wt
+!| prediction
+|-
+| wildtype
+| 164.21
+|align="center"| 1
+| no effect
+|-
+| ARG158GLN
+| 172.845
+|align="center"| 1.05
+|effect
+|-
+| GLN172HIS
+| 169.699
+|align="center"| 1.03
+|no effect
+|-
+| ARG243GLN
+| 152.987
+|align="center"| 0.93
+|effect
+|-
+| LEU255SER
+| 166.123
+|align="center"| 1.01
+|effect
+|-
+| MET276VAL
+| 164.472
+|align="center"| 1.00
+|effect
+|-
+| ARG297CYS
+| 166.646
+|align="center"| 1.01
+| no effect
+|-
+| ALA322GLY
+| 164.285
+|align="center"| 1.00
+|no effect
+|-
+| GLU330ASP
+| 171.68
+|align="center"| 1.04
+|effect
+|-
+| GLY337VAL
+| 164.304
+|align="center"| 1.00
+|no effect
+|-
+| ARG408TRP
+| 393.804
+|align="center"| 2.39
+| effect
+|-
+|}
+</div>
+</figtable>
+== foldX ==
+The other approach we used is foldX
+=== preparation ===
+We use the run.txt from the example at the test and adapt the list and individual list as its shown in the [[Journal structure based mutation analysis (PKU)#foldX|journal]].
+=== usage ===
+<source lang="bash">
+ scwrl -runfile run.txt
+</source>
+=== results ===
+As we already have results from the SCWRL run, we just compare those with the new results from foldX. The comparison with the sourounding structures is left for the reader as an exercise. The residue placed by SCWRL is colored in blue whereas the foldX residue is orange. THe surrounding structure is colored randomly by PyMol.
+<figure id="foldxstructcompare">
+[[File:structurecomparisonfoldxScwrl.png|thumb|500px|center|<caption>In this Figure, we show the differences in the structures which we got from both methods, where scwrl is colored in blue and foldX is colored in cyan. This is the only change and we expect it to have a very low effect on the activity of the protein, because no residues which are known for their importance are found in this region</caption>]]
+</figure>
+<center>
+<gallery widths=450px heights=250px perrow=2>
+<caption> structures for the SNPs derived from foldX </caption>
+File:pkufoldXmutation158.png|Mutation ARG158GLN: In this picture the changes between foldX and SCWRL are not big. The stuctural differences of the two residues result in the fact, that SCWRl includes H-atoms which are not included in foldX. Apart from that, the residues are twisted which results in a difference of the H-bonds. As one can see, SCWRL forms a bond (colored in blue) which is not present in the residue conformation in foldX.
+File:pkufoldXmutation172.png|Mutation GLN172HIS: Here the changes again are only a small change in the angle of the residue and the "missing" H-atom in the foldX-structure (orange). No other changes are obvious.
+File:pkufoldXmutation243.png|Mutation ARG243GLN: Due to the change in the conformation of the residues, the orange structure (foldX) can form a bond, whereas the blue structure is not able to form any polar bonds.
+File:pkufoldXmutation255.png|Mutation LEU255SER: In this case, in spite of the completely changed structures, both of the conformations are able to establish bonds to the same target, But as they are formed from a different angle, we expect this to change the structure through torsion of the molecules.
+File:pkufoldXmutation276.png|Mutation MET276VAL: As the structure derived from SCWRL (blue) can form the blue bond, which can not be formed in the structure from foldX (orange), the structure of SCWRL is probably quite different from the other one.
+File:pkufoldXmutation297.png|Mutation ARG297CYS: With the torsion of the angle of the two residues, they seem to have a quite different properties, but the changes seem to be rather minor if one has a look at the surroundings.
+File:pkufoldXmutation322.png|Mutation ALA322GLY: In this case there is no difference in the structures derived from both methods
+File:pkufoldXmutation330.png|Mutation GLU330ASP: Similar to the case ARG158GLU the structure which is proposed by SCWRL can build one more polar bond, than the corresponding structure.
+File:pkufoldXmutation337.png|Mutation GLY337VAL: Again the changes seem to be rather big, but there are no visible changes in the bonds or clashes
+File:pkufoldXmutation408.png|Mutation ARG408TRP: This is the only mutation, in which the two methods support completly different structures, which do not yield any clashes or bonds, which are possible in the one and not in the other.
+</gallery>
+</center>
+<figtable id="tab:foldxenergies">
+<center><caption>Comparison of all the energies for the mutations and wildtype for foldX </caption></center>
+<div style="float: center; width: 100%">
+{| align="center"
+!| Mutation
+!| energy
+!| energy mut/energy wt
+!| energy mut/energy relative wt
+!| prediction
+|-
+| wildtype
+| 14.00
+|align="center"| 1
+|align="center"| 1
+| no effect
+|-
+| ARG158GLN
+| 6.36
+|align="center"| 0.45
+|align="center"| 1.31
+| effect
+|-
+| GLN172HIS
+| 16.69
+|align="center"| 1.16
+|align="center"| 0.99
+|no effect
+|-
+| ARG243GLN
+| 6.15
+|align="center"| 0.43
+|align="center"| 0.72
+| effect
+|-
+| LEU255SER
+| 23.57
+|align="center"| 1.68
+|align="center"| 1.21
+|effect
+|-
+| MET276VAL
+| 21.99
+|align="center"| 1.57
+|align="center"| 1.06
+|effect
+|-
+| ARG297CYS
+| 20.31
+|align="center"| 1.45
+|align="center"| 1.01
+|no effect
+|-
+| ALA322GLY
+| 21.76
+|align="center"| 1.55
+|align="center"| 1.05
+|no effect
+|-
+| GLU330ASP
+| 19.65
+|align="center"| 1.40
+|align="center"| 1.02
+|effect
+|-
+| GLY337VAL
+| 24.01
+|align="center"| 1.71
+|align="center"| 1.10
+|effect
+|-
+| ARG408TRP
+| 34.36
+|align="center"| 2.45
+|align="center"| 1.58
+| effect
+|-
+|}
+</figtable>
+== minimise ==
+=== preparation ===
+To use minimise we have to clear the structure of hydrogen atoms and solvent which we do again using the [[repairPDB]] script.
+<source lang="bash">
+$ repairPDB 1J8U.pdb -noh -nosol > 1J8U_pure.pdb
+</source>
+=== usage ===
+Afterwards a little script is used to apply the minimization five times and each time using the output as input.
+*usage:
+<source lang="bash">
+$ runAllMinimiser.sh <filestem>
+</source>
+*[[Journal structure based mutation analysis (PKU)#runAllMinimiser.sh|script]]
+=== results ===
+Just to see the differences between the results of one run and several using the output again as input, we produce this little animated .gif, which is basicly all the results from the 18JU.pdb on till the fifth minimise result.
+<figure id="fig:helixminimise">
+[[File:1J8Uminimised.gif|center|500px|thumb|<caption>Superimposition of all structures derived from minimise for 1J8U which means the wildtype. For a better comparison first the wildtype is shown in blue. Then it will get transparent and the first result of minimize is shown in teal. Then the transparent wildtype disappears and the first result will get transparent and the second result will show. This continues until the final result which is red. Then the animation will start again.</caption>]]
+</figure>
+In <xr id="fig:helixminimise" /> one can see that minimise shifts the start of the helix further down in the picture, and once it reaches a certain point, the start of the helix is converted to a coiled region instead. A similar effect can be found if one compares sheets in the output of the different minimise runs. Here a twisting of the sheets takes place, which can bee seen in <xr id="fig:sheetminimise" />
+<figure id="fig:sheetminimise">
+[[File:1J8Usheetminimised.png|thumb|500px|center|<caption>Structure of 1J8U before (cyan) and after (red) minimise. The red structure is the result of five consecutive runs of minmise, and shows a twist in the orientation of the sheet.</caption>]]
+</figure>
+<figure id="fig:timecourseminimise">
+[[File:pku1J8Utimecourseminimise.png|thumb|400px|center|<caption>This picture shows the time course of the energy derived from minimise for 14 steps for the 1J8U-structure we got from the pdb and excluded the solvent</caption>]]
+</figure>
+As one can see in <xr id="fig:timecourseminimise" /> consecutive multiple minimise runs do not decrease the energy but increase it drastically. But the first two minimise steps reduce the energy, and therefore we want to compare the energyoutput from minimise to the two others found in <xr id="tab:foldxenergies" /> and <xr id="tab:scwrlenergies" />.
+<figtable id="tab:minimiseenergies">
+<center><caption>Comparison of all mutations with energies derived from minimise</caption>
+{| align="center" style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; border-width: 0 1px 1px 0"|mutation
+!style="border-style: solid; border-width: 0 1px 1px 0"|energy foldX
+!style="border-style: solid; border-width: 0 1px 1px 0"|energy foldX/energy wt
+!style="border-style: solid; border-width: 0 1px 1px 0"|energy scwwrl
+!style="border-style: solid; border-width: 0 1px 1px 0"|energy scwrl/energy wt
+!style="border-style: solid; border-width: 0 0 1px 0"|prediction
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"|wildtype
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7516.267118
+|style="border-style: solid; border-width: 0 1px 1px 0"|
+|style="border-style: solid; border-width: 0 1px 1px 0"|
+|style="border-style: solid; border-width: 0 1px 1px 0"|
+|style="border-style: solid; border-width: 0 0 1px 0"| no effect
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| ARG158GLN
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7564.267932
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"|1.00
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7400.825142
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| GLN172HIS
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7513.718344
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7375.087666
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| ARG243GLN
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7522.768865
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 1.00
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7476.255952
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| LEU255SER
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7522.768865
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"|1.00
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7476.255952
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| MET276VAL
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7502.369873
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7374.091542
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| ARG297CYS
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7616.286033
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 1.01
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7484.442763
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| ALA322GLY
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7509.307761
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7389.927555
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| GLU330ASP
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7494.936652
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7367.788205
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| GLY337VAL
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7504.533976
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.99
+|style="border-style: solid; border-width: 0 1px 1px 0"| -7380.216180
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"|0.98
+|-
+|style="border-style: solid; border-width: 0 1px 1px 0"| ARG408TRP
+|style="border-style: solid; border-width: 0 1px 1px 0"| -5289.743707
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.70
+|style="border-style: solid; border-width: 0 1px 1px 0"| -5438.301688
+|style="border-style: solid; border-width: 0 1px 1px 0" align="center"| 0.72
+|style="border-style: solid; border-width: 0 0 1px 0"| effect
+|-
+|}
+</figtable>
+TODO: this and maybe compare with the other weird energy outputs from SCWRL and foldX und dann halt irgendwie daraus die prediction für die einzelnen Mutationen ableiten
+== surface considerations ==
+As we only can get very little information from the minimise-results, we try to include the overall surface of the protein as well as the important sites into the prediction of the effectivity. In the following, mutants are colored red and wildtype residues green. The wildtype surface is shown in green and grey (for non mutant areas). In contrast the mutant surface will be shown as a mesh and in red color.
+<center>
+<gallery widths=450px heights=250px perrow=2>
+<caption> structures for the SNPs derived from foldX </caption>
+File:pkusurfacemutation158.png|Mutation ARG158GLN: As the arginine is a rather long residue, which sticks out of the protein in most cases, here we can find some kind of small hill arising in green, where the red mesh stays rather flat due to the dens structure of glutamine
+File:pkusurfacemutation172.png|Mutation GLN172HIS: As the plain structures of the mutant in red and the wildtype residue in green are quite different the result in surface is not big, the only thing which is changing, is that the green bulge is now in a silightly different angle.
+File:pkusurfacemutation243.png|Mutation ARG243GLN: In this picture we see the big differences which come with the change of the amino acids. As the normal surface is kind of a small tunnel on the upper left, with the change to glutamine this tunnel would widen (see red mesh) and a new hole on the right would appear, which is not present in the green surface (wildtype)
+File:pkusurfacemutation255.png|Mutation LEU255SER: Like in the picture before (ARG243GLN) with the mutation comes a bigger hole in the structure, which is due to the smaller size of the new amino acid
+File:pkusurfacemutation276.png|Mutation MET276VAL: This mutation yields big changes in the structure, as the wildtype (green) formed a slightly descending hyperplane, the red mesh shows a bulge outof the structure which is rising where the wildtype was falling.
+File:pkusurfacemutation297.png|Mutation ARG297CYS: With the rather long arginine substituted with a in comparison small cysteine, the apex which was formed by arginine disappears and instead forms a small dent
+File:pkusurfacemutation322.png|Mutation ALA322GLY: Again due to the change of a bigger residue to a smaller one, the hole in the protein gets widened
+File:pkusurfacemutation330.png|Mutation GLU330ASP: Like in ALA322GLY the mutant residue (red) widens the space in the protein
+File:pkusurfacemutation337.png|Mutation GLY337VAL: As the mutant residue is bulkier than the original one, the peak which was present in the wildtype (green) also get bulkier
+File:pkusurfacemutation408.png|Mutation ARG408TRP: although the amino acid length between arginine and tryptophan is pretty much the same, their width is different. Due to this fact, a new rift in the protein opens and the overall conformation of this part changes, as one can see with the large mesh around the red mutant residue
+</gallery>
+</center>
+<figtable id="tab:scwrlenergies">
+<center><caption>Comparison of the surface changes with respect to known binding sites and location </caption></center>
+<div style="float: center; width: 100%">
+{| align="center"
+!| Mutation
+!| surface changes
+!| bindingsite
+!| prediction
+|-
+| ARG158GLN
+| mild to big
+| no
+| effect
+|-
+| GLN172HIS
+| mild
+| no
+| no effect
+|-
+| ARG243GLN
+| big
+| no
+| effect
+|-
+| LEU255SER
+| mild
+| yes
+| effect
+|-
+| MET276VAL
+| big
+| no
+| no effect
+|-
+| ARG297CYS
+| big
+| no
+| effect
+|-
+| ALA322GLY
+| mild
+| yes
+| effect
+|-
+| GLU330ASP
+| mild
+| yes
+| effect
+|-
+| GLY337VAL
+| big
+| no
+| effect
+|-
+| ARG408TRP
+| huge
+| no
+| effect
+|-
+|}
+</div>
+</figtable>
+==Gromacs==
+We choose the scwrl structures as input, since there is little difference to observe so far, but scwrl will be used in next week's task and results will be more comparable that way. The provided fetchPDB-script just wgets the gzipped PDBfile for a given PDB identifier, but was not used, since we already had our input structures. To extract the protein alone from the PDB-file, we used repairPDB with the -jprot option. repairPDB provides a number of usefull options, e.g. it extracts or removes DNA, crystal water, selects individual chains or renumbers the sequence.
+The minimization preparation and runs were performed with [[Journal_structure_based_mutation_analysis_(PKU)#prepare for Gromacs|this script]]. There is also a short script to run g_energy from the .edr-files [[Journal_structure_based_mutation_analysis_(PKU)#run g_energy|here]]. We initially choose the forcefields TIP3P for water, AMBER03 (option 1 in the interactive menu), CHARMM27 (option 8) and GROMOS96 53a6 (option 13) for the protein, since they are amongst the most popular, but run into troubles with GROMOS and replaced it with OPLS-AA (option 14). All of them work with our chosen water model TIP3P. We used the provided standard configuration  file for gromacs, described [[MDP condiguration (PKU)|elsewhere]].
+===Results===
+====Runtime====
+For most mutants and most forcefields the minimization converges between step 250 and 300 and calculation stops then, so there is little sense in starting with a value of more then say 1000 for nsteps. If the energy does not converge by then, we'd suspect some error in the input. We give a few runtimes for the wildtype and the AMBER03 forcefield in <xr id="fig:runtime"/>, performed on the lrz grid. The maximum runtime we observed was around 50 seconds (not shown), the average runtime for a minimization is around 30 seconds. Since the calculation of every step requires the same amount of work, the expected runtime behaviour until convergence is perfectly linear in ''nsteps'' plus a constant for initialization. The provided datapoints confirm this nicely.
+<figure id="fig:runtime">
+[[File:TimedRuns.png|400px|thumb|right|Runtime for different values for nsteps of minimization with Gromacs, using the AMBER03 forcefield and our wildtype 1J8U.]]
+</figure>
+====Energies====
+We show the curve of the angle, bond and potential energy during the wildtype minimization for all three forcefields in <xr id="fig:1J8U_energies"/>. In all cases, the energy goes down steeply at first. While the bond energy does not change much after that and potential energy slowly converges, the angle energy rises during optimization until it also converges at a level slightly higher than its initial lowest point. This is as expected, since the total energy is optimized by adjusting the conformation of the protein. The mutants behave qualitatively similar, plots will be available [[Mutation Minimization Plots (PKU)|here]], once they are uploaded.
+The tables <xr id="tab:gromacs_energies_1J8U"/> to <xr id="tab:gromacs_energies_mut408"/> show the energies of angle, bond and the potential energy of the wildtype and the ten mutants. In brackets, the difference to the wildtype is given. The bond energy comprises a small part of the total energy and changes not much in the mutants (the largest change in ARG158GLN is 2.6%). Neither direction nor amplitude of the energy changes seem to correlate with malignance of the mutations directly.
+For the AMBER force field the average change in potential energy for the mutants is +304 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +139 kJ/mol. On the other hand, the smallest change in energy is observed in a disease causing, the GLY337VAL mutation. All but one disease causing mutation increase the potential energy, but two of the three benign ones do the same.
+So far, we observe that smaller changes in energy indicate a less severe impact on the structure, but the prediction value appears limited, since the false positives number (benign mutations thought to be malign) is high. Setting a cut-off of the potential energy change to 100 kJ/mol would correctly classify five disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 70%.
+For the CHARMM force field the average change in potential energy for the mutants is +712 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +101 kJ/mol. The largest change in a benign mutation is only +160.53 kJ/mol and there are four extreme changes above 900 kJ/mol in the malign mutations. Setting a cut-off of the potential energy change to 100 kJ/mol would correctly classify six disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 80%.
+For the OPLS-AA force field the average change in potential energy for the mutants is +151 kJ/mol, the average change in benign or hyperphenylalanemia causing mutations is only +57 kJ/mol. The largest change in a benign mutation is +147.33 kJ/mol and there are three larger changes above 100 kJ/mol in the malign mutations, but also one change of +147 kJ/mol in an benign mutation. Setting a cut-off of the potential energy change to 50 kJ/mol would correctly classify five disease causing mutations above and two benign mutations below this threshold, giving an accuracy of 70%.
+In all force fields, the largest change is caused by the malign ARG408TRP mutation, a [[Predicting_the_Effect_of_SNPs_(PKU)#ARG408TRP|visibly extreme disruption]] of the structure. Rather surprising is the large change in the benign GLN172HIS, that is located in a coiled region at the outside of PheOh and was one of the [[Predicting_the_Effect_of_SNPs_(PKU)#GLN172HIS|easier cases]] in last week's prediction task.
+<figtable id="fig:1J8U_energies">
+<caption>Course of the energies during minimization of 1J8U with gromacs.</caption>
+{| style="border-style: solid; border-width: 1px"
+|[[File:1J8U nh amber03 angle.png|200px|thumb|left|Energie of the angles in 1J8U in the AMBER03 forcefield.]]
+|[[File:1J8U nh amber03 bonds.png|200px|thumb|left|Energie of the bonds in 1J8U in the AMBER03 forcefield.]]
+|[[File:1J8U nh amber03 potential.png|200px|thumb|left|Potential energy in 1J8U in the AMBER03 forcefield.]]
+|-
+|<!--[[File:1J8U nh charmm27 angle.png|200px|thumb|left|Energie of the angles in 1J8U in the CHARMM27 forcefield.]]-->
+|[[File:1J8U nh charmm27 bonds.png|200px|thumb|left|Energie of the bonds in 1J8U in the CHARMM27 forcefield.]]
+|[[File:1J8U nh charmm27 potential.png|200px|thumb|left|Potential energy in 1J8U in the CHARMM27 forcefield.]]
+|-
+|[[File:1J8U nh oplsaa angle.png|200px|thumb|left|Energie of the angles in 1J8U in the OPLS-AA forcefield.]]
+|[[File:1J8U nh oplsaa bonds.png|200px|thumb|left|Energie of the bonds in 1J8U in the OPLS-AA forcefield.]]
+|[[File:1J8U nh oplsaa potential.png|200px|thumb|left|Potential energy in 1J8U in the OPLS-AA forcefield.]]
+|-
+|}
+</figtable>
+<figtable id="tab:gromacs_energies_1J8U">
+<caption>Energies after Gromacs minimization of the wildtype.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2403.47 (Δ 0.00)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3333.34(Δ 0.00)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2092.75 (Δ 0.00)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 522.31 (Δ 0.00)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1162.70(Δ 0.00)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 545.17 (Δ 0.00)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37536.96 (Δ 0.00)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40392.46(Δ 0.00)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65323.58 (Δ 0.00)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut158">
+<caption>Energies after Gromacs minimization of the disease causing ARG158GLN mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2387.31 (Δ -16.17)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3287.85(Δ -45.48)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2087.86 (Δ -4.90)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 509.25 (Δ -13.06)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1138.40(Δ -24.30)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 543.58 (Δ -1.59)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37171.73 (Δ 365.23)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -39342.72(Δ 1049.75)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65223.57 (Δ 100.01)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut172">
+<caption>Energies after Gromacs minimization of the benign GLN172HIS mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2479.53 (Δ 76.06)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3316.94(Δ -16.40)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2106.61 (Δ 13.86)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 522.68 (Δ 0.37)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1154.75(Δ -7.95)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 546.84 (Δ 1.67)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37163.15 (Δ 373.82)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40231.94(Δ 160.53)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65176.25 (Δ 147.33)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut243">
+<caption>Energies after Gromacs minimization of the disease causing ARG243GLN mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2398.46 (Δ -5.01)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3303.89(Δ -29.45)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2086.91 (Δ -5.84)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 517.89 (Δ -4.43)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1144.20(Δ -18.50)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 542.42 (Δ -2.76)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37333.11 (Δ 203.86)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -39440.42(Δ 952.05)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65231.15 (Δ 92.43)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut255">
+<caption>Energies after Gromacs minimization of the disease causing LEU255SER mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2415.15 (Δ 11.68)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3311.82(Δ -21.52)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2092.62 (Δ -0.13)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 530.22 (Δ 7.91)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1150.95(Δ -11.75)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 546.59 (Δ 1.41)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37613.71 (Δ -76.75)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40227.79(Δ 164.67)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65323.13 (Δ 0.45)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut276">
+<caption>Energies after Gromacs minimization of the benign MET276VAL mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2402.78 (Δ -0.70)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3315.16(Δ -18.18)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2094.12 (Δ 1.37)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 522.62 (Δ 0.31)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1153.45(Δ -9.25)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 548.90 (Δ 3.72)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37460.79 (Δ 76.18)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40308.56(Δ 83.90)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65297.64 (Δ 25.95)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut297">
+<caption>Energies after Gromacs minimization of the disease causing ARG297CYS mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2404.12 (Δ 0.65)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3295.31(Δ -38.03)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2088.02 (Δ -4.73)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 521.46 (Δ -0.86)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1140.64(Δ -22.06)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 547.49 (Δ 2.32)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -36943.84 (Δ 593.13)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -39121.65(Δ 1270.81)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65074.67 (Δ 248.91)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut322">
+<caption>Energies after Gromacs minimization of the hyperphenylalaninemia causing ALA322GLY mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2409.61 (Δ 6.13)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3303.45(Δ -29.88)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2091.98 (Δ -0.77)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 528.05 (Δ 5.74)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1147.60(Δ -15.10)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 547.21 (Δ 2.03)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37567.33 (Δ -30.36)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40334.32(Δ 58.14)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65325.79 (Δ -2.21)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut330">
+<caption>Energies after Gromacs minimization of the disease causing GLU330ASP mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2408.24 (Δ 4.77)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3337.37(Δ 4.03)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2097.44 (Δ 4.69)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 522.71 (Δ 0.39)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1162.17(Δ -0.53)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 544.42 (Δ -0.75)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37373.77 (Δ 163.19)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40487.35(Δ -94.89)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65280.02 (Δ 43.57)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut337">
+<caption>Energies after Gromacs minimization of the disease causing GLY337VAL mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2415.06 (Δ 11.58)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3332.88(Δ -0.46)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2105.72 (Δ 12.97)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 526.54 (Δ 4.23)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1156.77(Δ -5.93)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 548.96 (Δ 3.79)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -37518.37 (Δ 18.60)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -40272.05(Δ 120.41)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -65273.33 (Δ 50.25)
+|-
+|}
+ </figtable>
+<figtable id="tab:gromacs_energies_mut408">
+<caption>Energies after Gromacs minimization of the disease causing ARG408TRP mutation.</caption>
+{| style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; padding-left:5px; padding-right:5px; border-color: #000; padding: 0"
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Type
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Amber03
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Charmm27
+!style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | Oplsaa
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Angle (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 2430.32 (Δ 26.85)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | <!--3331.13(Δ -2.21)-->
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 2110.42 (Δ 17.67)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Bonds (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 524.16 (Δ 1.84)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | 1150.43(Δ -12.26)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | 546.33 (Δ 1.15)
+|-
+|style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | Potential (kJ/mol)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -36674.55 (Δ 862.41)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 1px 1px 0;" | -38873.69(Δ 1518.78)
+| style="border-style: solid; padding-left:5px; padding-right:5px; border-width: 0 0px 1px 0;" | -64802.38 (Δ 521.21)
+|-
+|}
+ </figtable>
+[[Category: Phenylketonuria 2012]]

Difference between revisions of "Structure-based mutation analysis (PKU)"

Latest revision as of 11:49, 29 August 2012

Contents

Short task description

Finding the right structure

1J8U

Mutations

ARG158GLN

GLN172HIS

ARG243GLN

LEU255SER

MET276VAL

ARG297CYS

ALA322GLY

GLU330ASP

GLY337VAL

ARG408TRP

SCWRL

preparations

usage

results

foldX

preparation

usage

results

minimise

preparation

usage

results

surface considerations

Gromacs

Results

Runtime

Energies

Navigation menu

Search