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== Structure selection == The following table presents the PDB structures for BCKDHA to date: {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !PDB id !resolution [Å] !R-factor !coverage !ph-value |- |1DTW||2.70||0.224||||7.5* |- |1OLS||1.85||0.172||||5.5 |- |1OLU||1.90||0.161||||5.5 |- |1OLX||2.25||0.161||||5.5 |- |1U5B||1.83||0.156||||5.8 |- |1V11||1.95||0.139*||||5.5 |- |1V16||1.90||0.132*||||5.5 |- |1V1M||2.00||0.130*||||5.5 |- |1V1R||1.80||0.158||||5.5 |- |1WCI||1.84||0.149||||5.5 |- |1X7W||1.73||0.148||||5.8 |- |1X7X||2.10||0.149||||5.8 |- |1X7Y||1.57||0.150||||5.8 |- |1X7Z||1.72||0.154||||5.8 |- |1X80||2.00||0.161||||5.8 |- |2BEU||1.89||0.171||||5.5 |- |2BEV||1.80||0.139||||5.5 |- |2BEW||1.79||0.147||||5.5 |- |2BFB||1.77||0.145||||5.5 |- |2BFC||1.64||0.144||||5.5 |- |2BFD||1.39*||0.150||||5.5 |- |2BFE||1.69||0.150||||5.5 |- |2BFF||1.46||0.150||||5.5 |- |2J9F||1.88||0.171||||5.5 |} We could not use any of the PDB structures for BCKDHA because all of them had gaps in the secondary structure which means that some residues were missing. So we took the structure which has the less gaps: '''1U5B'''<br> *resultion: 1.83<br> *R-factor: 0.156<br> *ph-value: 5.8 This structure has to be modified with some programms to close the gaps. Additionally the first residues which are in BCKDHA misses in 1U5B thats why the startposition corresponds to position 6 of the BCKDHA sequence. As we can see non of the values correspond to the demand because it was ask for an structure which has a very small R-factor, a pH of 7.4 and a high resolution. == Mapping of the mutations on the crystal structure == [[File:BCKDHA_mutations_activeSites.png|thumb|400px|center|Structure of BCKDHA. Violet: mutations, orange: thiamine pyrophosphate binding sites, yellow: metal binding sites.]] Showing hydrogen bonds with Pymol: A -> find -> polar contacts -> within selection ;M82L {|class="centered" |[[File:BCKDHA_Wt37.png|thumb|150px|Hydrogen Bonds for methionine on pos 82 in the wild type structure]] |[[File:BCKDHA_Mut37.png|thumb|150px|Hydrogen Bonds for leucine on pos 82 in the mutated type structure]] |} ;Q125E {|class="centered" |[[File:BCKDHA_Wt80.png|thumb|150px|Hydrogen Bonds for glutamine on pos 125 in the wild type structure]] |[[File:BCKDHA_Mut80.png|thumb|150px|Hydrogen Bonds for glutamic acid on pos 125 in the mutated type structure]] |} ;Y166N {|class="centered" |[[File:BCKDHA_Wt121.png|thumb|150px|Hydrogen Bonds for tyrosine on pos 166 in the wild type structure]] |[[File:BCKDHA_Mut121.png|thumb|150px|Hydrogen Bonds for asparagine on pos 166 in the mutated type structure]] |} ;G249S {|class="centered" |[[File:BCKDHA_Wt204.png|thumb|150px|Hydrogen Bonds for glycine 249 in the wild type structure]] |[[File:BCKDHA_Mut204.png|thumb|150px|Hydrogen Bonds for serine on pos 249 in the mutated type structure]] |} ;C264W {|class="centered" |[[File:BCKDHA_Wt219.png|thumb|150px|Hydrogen Bonds for cystein on pos 264 in the wild type structure]] |[[File:BCKDHA_Mut219.png|thumb|150px|Hydrogen Bonds for tryptophan on pos 264 in the mutated type structure]] |} ;R265W {|class="centered" |[[File:BCKDHA_Wt220.png|thumb|150px|Hydrogen Bonds for arginine on pos 265 in the wild type structure]] |[[File:BCKDHA_Mut220.png|thumb|150px|Hydrogen Bonds for tryptophane on pos 265 in the mutated type structure]] |} ;I326T {|class="centered" |[[File:BCKDHA_Wt281.png|thumb|150px|Hydrogen Bonds for isoleucine on pos 326 in the wild type structure]] |[[File:BCKDHA_Mut281.png|thumb|150px|Hydrogen Bonds for threonine on pos 326 in the mutated type structure]] |} ;F409C {|class="centered" |[[File:BCKDHA_Wt364.png|thumb|150px|Hydrogen Bonds for phenylalanine on pos 409 in the wild type structure]] |[[File:BCKDHA_Mut364.png|thumb|150px|Hydrogen Bonds for cysteine on pos 409 in the mutated type structure]] |} ;Y438N {|class="centered" |[[File:BCKDHA_Wt393.png|thumb|150px|Hydrogen Bonds for tyrosine on pos 438 in the wild type structure]] |[[File:BCKDHA_Mut393.png|thumb|150px|Hydrogen Bonds for asparagine on pos 438 in the mutated type structure]] |} == Comparison energies == === SCWRL === Before we could use SCWRL we first had to get the sequence of our model: repairPDB bckdha.pdb -seq >> bckdha.seq When we have the sequence we have to make one file for each mutation. In these files we copied the bckdha.seq and changed the sequence to lower case letters. Then we add the mutation in an upper case letter. To run SCWRL we used the command: scwrl -i bckdha.pdb -s mutation1.seq -o mutation1Model.pdb '''Total minimal energy of the graph''' {|border="1" !Position !Energy |- |M82L||642.213 |- |Q125E||616.85 |- |Y166N||616.293 |- |G249S||633.378 |- |C264W||805.257 |- |R265W||710.647 |- |I326T||619.424 |- |F409C||617.305 |- |Y438N ||615.951 |} === foldX === To use foldX we first build a [[runscript for foldx |runscript]]. It is important to change values of <Temperature> and <pH> to the values of the used protein. These values can be found on the [http://www.pdb.org pdb side] . Additionally we had to create one file with all PDB Ids each in a new line (list.txt). We used the command <code> Foldx -runfile run.txt > Stout.txt </code> to run the programm.<br> {|border="1" ! !total energy !difference |- |wildtype||401.00||0 |- |M82L||437.88||-36.88 |- |Q125E||431.77||-30.77 |- |Y166N||432.24||-31.24 |- |G249S||432.22||-31.22 |- |C264W||488.43||-87.43 |- |R265W||460.43||-59.43 |- |I326T||432.94||-31.94 |- |F409C||433.33||-32.33 |- |Y438N||431.56||-30.56 |} After using foldx we have the total energy for the wiltype protein and for each mutation. The value of the wildtype protein is 401.00 which is already a high value. This means that the protein is quite instabile. To find out which mutation has a high influence on the protein we look at the energies and especially on the difference between the energy of the mutated protein and the wildtype protein. All of the mutated proteins have a much higher energy than the unmutated protein which means that these proteins are less stable. We can see in the table that the proteins can be divided into two groups. The first group has an energy difference of about 31 and the other group has a much higher difference. === Minimise === It is important to remove the hydrogens and water before using the programm. For this we used the new version of repairPDB of the virtualbox. The programm can be started with the command: <code>repairPDB bckdha.pdb -nosol out.pdb > Stout.txt </code> <br> It is also possible to use the old version but then the command is: <code>repairPDB bckdha.pdb -nosol -noh out.pdb > Stout.txt </code> <br> It is useful to save the output in a file because it includes the energy. {|border="1" ! !total energy !difference |- |wildtype|| -2485.452755|| 0 |- |M82L|| -4253.174790||1767.722015 |- |Q125E|| -4080.989512|| 1595.536757 |- |Y166N|| -4354.495238|| 1869.042483 |- |G249S|| -4280.043000||1794.590245 |- |C264W|| -3745.313620||1259.860865 |- |R265W|| -3989.790625||1504.33787 |- |I326T|| -4317.105618||1831.652863 |- |F409C|| -4358.528143||1873.075388 |- |Y438N|| -4339.778964||1854.326209 |} Minimise calculates the energy for a mutation by building a new model for each mutation. And then it calculates the energy for the whole mutated model. To find out if there is a difference between the wildtype and the model that is calculated by Minimise. The aim by comparing the mutated models with the wildtype is to find out if there is a structural change caused by a mutation. We superposed each mutated protein with the wildtype and focused on the mutated position. In the pictures there are always the superposed structures. In the wildtype pictures the structure of the unmutated residue is bold and in the mutated pictures the structure of the mutated residue is bold. So we can compare the two pictures to see if there is a change in the structure caused by the mutation on this residue. {|border="1" !mutation !wildtype structure !mutated structure |- |M82L||[[File:Wildtype1_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation1_BCKDHAMinimise.png|thumb|mutation M82L]] |- |Q125E||[[File:Wildtype2_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation2_BCKDHAMinimise.png|thumb|mutation Q125E]] |- |Y166N||[[File:Wildtype3_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation3_BCKDHAMinimise.png|thumb|mutation Y166N]] |- |G249S||[[File:Wildtype4_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation4_BCKDHAMinimise.png|thumb|mutation G249S]] |- |C264W||[[File:Wildtype5_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation5_BCKDHAMinimise.png|thumb|mutation C264W]] |- |R265W||[[File:Wildtype6_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation6_BCKDHAMinimise.png|thumb|mutation R265W]] |- |I326T||[[File:Wildtype7_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation7_BCKDHAMinimise.png|thumb|mutation I326T]] |- |F409C||[[File:Wildtype8_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation8_BCKDHAMinimise.png|thumb|mutation F409C]] |- |Y438N||[[File:Wildtype9_BCKDHAMinimise.png|thumb|wildtype]]||[[File:Mutation9_BCKDHAMinimise.png|thumb|mutation Y438N]] |} == Gromacs == The first part describes general background information for gromacs as well as how to run those programs. The second part contains the result description and analysis. === General === ====1. fetchpdb==== The fetch-pdb script first checks, if it was called with an valid PDB-id. If the entered PDB code has 4letters, the script tries to download the pdb-file from the server. The successfully downloaded folder gets unzipped and everything except the actual pdb file is removed. ====2. repairPDB==== repairPDB bckdha_mod.pdb -noh -nosol > bckdha_clean.pdb ====3. SCWRL ==== scwrl -i bckdha_mod.pdb -s extractedPDB.seq -o bckdha_scwrl.pdb pdb including HEATOMS ====4.pdb2gmx==== use clean pdb without HEATOMS pdb2gmx -f bckdha_clean.pdb -o bckdha.gro -p bckdha.top -water tip3p -ff amber03 ====5. MDP==== <!-- <code> title = PBSA minimization in vacuum cpp = /usr/bin/cpp define = -DFLEXIBLE -DPOSRES implicit_solvent = GBSA integrator = steep emtol = 1.0 nsteps = 500 nstenergy = 1 energygrps = System ns_type = grid coulombtype = cut-off rcoulomb = 1.0 rvdw = 1.0 constraints = none pbc = no </code {| |integrator|| a steepest descent algorithm for energy minimization. |- |emtol||tolerance for steep integrator:the minimization is converged when the maximum force is smaller than this value |- |nsteps ||maximum number of steps to integrate or minimize, -1 is no maximum |- |nstenergy ||frequency to write energies to energy file (last energies are always written) |- |energygrps ||groups to write to energy files |- |ns_type|| |- |coulombtype |- |rcoulomb |- |rvdw |- |constraints |- |pbc --> ====6. grompp==== grompp -v -f MDP_bckdha.mdp -c bckdha.gro -p bckdha.top -o bckdha.tpr ====7. System Minimization ==== mdrun -v -deffnm bckdha 2> mdrun_out.txt ====8. Analyzation==== g_energy -f bckdha.edr -o energy_1.xvg === Analysis === ====Wildtype analysis: nsteps vs time ==== {| !steps !time (real) [s] |- |50 ||8.074 |- |100 ||10.362 |- |500 ||6.156 |- |1000||15.240 |- |5000||4.231 |} ==== Wildtype analysis: force fields==== The different force fields chosen for this task were: *AMBER03 [[File:Energy_bckdha_amber03.png|thumb|100px|right|GROMACS Energy for the AMBER03 forcefield using the wildtype bckdha structure.]] *CHARMM27 [[File:Energy_bckdha_charmm27.png|thumb|100px|right|GROMACS Energy for the CHARMM27 forcefield using the wildtype bckdha structure.]] *OPLS-AA [[File:Energy_bckdha_opls.png|thumb|100px|right|GROMACS Energy for the OPLS-AA forcefield using the wildtype bckdha structure.]] '''Bond Analysis''' {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Force Field !Average !Err. Est. !RMSD !Tot-Drift (kJ/mol) |- |AMBER03 ||3072.83||2200||-nan||-13100.2 |- |CHARMM25 ||3180.46||1700||7382.72||-9958.05 |- |OPLS ||2780.55||2100||-nan||-11542.6 |} '''Angle Analysis''' {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Force Field !Average !Err. Est. !RMSD !Tot-Drift (kJ/mol) |- |AMBER03 ||3616.97||230||-nan||-1295.57 |- |CHARMM25 ||5018.38||490||1646.81||-2783.35 |- |OPLS ||3271.23||340||-nan||-1889.98 |} '''Potential Analysis''' {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Force Field !Average !Err. Est. !RMSD !Tot-Drift (kJ/mol) |- |AMBER03 ||2.67001e+07||2.6e+07||-nan||-1.60382e+08 |- |CHARMM25 ||487.479||97199.742||673.043 |- |OPLS ||2.38353e+07||2.4e+07||-nan||-1.39932e+08 |} === Mutation analysis === ==== M82L ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 2518.71 || 1700||6337.97||-10023.3 |- |Angle || 3642.41||270|| 638.624||-1479.34 |- |Potential || 5.16e+06 ||5.1e+06||7.47e+07||-3.13e+07 |} ==== Q125E ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 2519.85 ||1700||6351.32||-10027.5 |- |Angle || 3626.21 ||260|| 618.433||-1418.24 |- |Potential || 5.23e+06|| 5.2e+06||7.5e+07||-3.17e+07 |} ==== Y166N ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 3029.19||2200||-nan||-12529.5 |- |Angle || 3654.58||280||-nan||-1486.71 |- |Potential || 7.95e+06||7.8e+06||-nan||-4.67e+07 |} ==== G249S ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 2775.97||2000||6761.45||-11375.2 |- |Angle || 3682.24||300||670.885||-1625.24 |- |Potential || 5.96e+06||5.0e+06||8.02e+07||-3.61e+07 |} ==== C264W ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 3186.75||2300||-nan||-13603.2 |- |Angle || 3831.06||370||-nan||-2070.89 |- |Potential || 3.41e+07||3.3e+07||-nan||-2.03e+08 |} ==== R265W ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 2473.43 ||1700||6385.14 || -9741.04 |- |Angle || 3726.4 ||330||827.187 || 1803.54 |- |Potential || 5.36e+06 || 5.3e+06|| 7.68e+07||-3.26e+07 |} ==== I326T ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 3214.03||2300||7364.47||-13490.1 |- |Angle || 3738.44||310||698.943||-1792.01 |- |Potential || 7.29e+06||6.9e+06||8.86e+07||-4.38e+07 |} ==== F409C ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 2341.69||1600||6048.14||-9087.07 |- |Angle || 3597.89||240||594.267||-1309.54 |- |Potential || 4.68e+06||4.7e+06||7.12e+07||-2.85e+07 |} ==== Y438N ==== {|border="1" style="border-spacing:0" align="center" cellpadding="3" cellspacing="3" !Energy !Average !Err.Est !RMSD !Tot-Drift (kJ/mol) |- |Bond || 3141.2||2300||-nan||-13216.1 |- |Angle || 3672.66||290||-nan||-1550.04 |- |Potential || 8.33e+06||8.1e+06||-nan||-4.94e+07 |} == Links == go back to [[Maple syrup urine disease]] main page go back to Task 6 [[Sequence-based mutation analysis_BCKDHA|Sequence based mutation analysis]] go to [[Reference Sequence BCKDHA]]

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