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Step 2

The PKA system is simple enough that there is no need to split up the PDB model file from Step 1 into several pieces. The modified model file is:

---
Label: CAMP-Dependent Protein Kinase (A and I chains only with water)
Entities:
- Label: A
Components: [LYS.8, GLY.9, SER.10, GLU.11, GLN.12, GLU.13, SER.14, VAL.15, LYS.16,
GLU.17, PHE.18, LEU.19, ALA.20, LYS.21, ALA.22, LYS.23, GLU.24, ASP.25, PHE.26,
LEU.27, LYS.28, LYS.29, TRP.30, GLU.31, ASN.32, PRO.33, ALA.34, GLN.35, ASN.36,
THR.37, ALA.38, HIS.39, LEU.40, ASP.41, GLN.42, PHE.43, GLU.44, ARG.45, ILE.46,
LYS.47, THR.48, LEU.49, GLY.50, THR.51, GLY.52, SER.53, PHE.54, GLY.55, ARG.56,
VAL.57, MET.58, LEU.59, VAL.60, LYS.61, HIS.62, LYS.63, GLU.64, THR.65, GLY.66,
ASN.67, HIS.68, PHE.69, ALA.70, MET.71, LYS.72, ILE.73, LEU.74, ASP.75, LYS.76,
GLN.77, LYS.78, VAL.79, VAL.80, LYS.81, LEU.82, LYS.83, GLN.84, ILE.85, GLU.86,
HIS.87, THR.88, LEU.89, ASN.90, GLU.91, LYS.92, ARG.93, ILE.94, LEU.95, GLN.96,
ALA.97, VAL.98, ASN.99, PHE.100, PRO.101, PHE.102, LEU.103, VAL.104, LYS.105,
LEU.106, GLU.107, TYR.108, SER.109, PHE.110, LYS.111, ASP.112, ASN.113, SER.114,
ASN.115, LEU.116, TYR.117, MET.118, VAL.119, MET.120, GLU.121, TYR.122, VAL.123,
PRO.124, GLY.125, GLY.126, GLU.127, MET.128, PHE.129, SER.130, HIS.131, LEU.132,
ARG.133, ARG.134, ILE.135, GLY.136, ARG.137, PHE.138, SER.139, GLU.140, PRO.141,
HIS.142, ALA.143, ARG.144, PHE.145, TYR.146, ALA.147, ALA.148, GLN.149, ILE.150,
VAL.151, LEU.152, THR.153, PHE.154, GLU.155, TYR.156, LEU.157, HIS.158, SER.159,
LEU.160, ASP.161, LEU.162, ILE.163, TYR.164, ARG.165, ASP.166, LEU.167, LYS.168,
PRO.169, GLU.170, ASN.171, LEU.172, LEU.173, ILE.174, ASP.175, GLN.176, GLN.177,
GLY.178, TYR.179, ILE.180, GLN.181, VAL.182, THR.183, ASP.184, PHE.185, GLY.186,
PHE.187, ALA.188, LYS.189, ARG.190, VAL.191, LYS.192, GLY.193, ARG.194, THR.195,
TRP.196, TPO.197, LEU.198, CYS.199, GLY.200, THR.201, PRO.202, GLU.203, TYR.204,
LEU.205, ALA.206, PRO.207, GLU.208, ILE.209, ILE.210, LEU.211, SER.212, LYS.213,
GLY.214, TYR.215, ASN.216, LYS.217, ALA.218, VAL.219, ASP.220, TRP.221, TRP.222,
ALA.223, LEU.224, GLY.225, VAL.226, LEU.227, ILE.228, TYR.229, GLU.230, MET.231,
ALA.232, ALA.233, GLY.234, TYR.235, PRO.236, PRO.237, PHE.238, PHE.239, ALA.240,
ASP.241, GLN.242, PRO.243, ILE.244, GLN.245, ILE.246, TYR.247, GLU.248, LYS.249,
ILE.250, VAL.251, SER.252, GLY.253, LYS.254, VAL.255, ARG.256, PHE.257, PRO.258,
SER.259, HIS.260, PHE.261, SER.262, SER.263, ASP.264, LEU.265, LYS.266, ASP.267,
LEU.268, LEU.269, ARG.270, ASN.271, LEU.272, LEU.273, GLN.274, VAL.275, ASP.276,
LEU.277, THR.278, LYS.279, ARG.280, PHE.281, GLY.282, ASN.283, LEU.284, LYS.285,
ASP.286, GLY.287, VAL.288, ASN.289, ASP.290, ILE.291, LYS.292, ASN.293, HIS.294,
LYS.295, TRP.296, PHE.297, ALA.298, THR.299, THR.300, ASP.301, TRP.302, ILE.303,
ALA.304, ILE.305, TYR.306, GLN.307, ARG.308, LYS.309, VAL.310, GLU.311, ALA.312,
PRO.313, PHE.314, ILE.315, PRO.316, LYS.317, PHE.318, LYS.319, GLY.320, PRO.321,
GLY.322, ASP.323, THR.324, SER.325, ASN.326, PHE.327, ASP.328, ASP.329, TYR.330,
GLU.331, GLU.332, GLU.333, GLU.334, ILE.335, ARG.336, VAL.337, SER.338, ILE.339,
ASN.340, GLU.341, LYS.342, CYS.343, GLY.344, LYS.345, GLU.346, PHE.347, SER.348,
GLU.349, PHE.350, MG.401, MG.402, ATP.400, HOH.404, HOH.405, HOH.406,
HOH.407, HOH.408, HOH.409, HOH.410, HOH.411, HOH.412, HOH.413, HOH.414, HOH.415,
HOH.416, HOH.417, HOH.418, HOH.419, HOH.420, HOH.421, HOH.422, HOH.423, HOH.424,
HOH.425, HOH.426, HOH.427, HOH.428, HOH.429, HOH.430, HOH.431, HOH.432, HOH.433,
HOH.434, HOH.435, HOH.436, HOH.437, HOH.438, HOH.439, HOH.440, HOH.441, HOH.442,
HOH.443, HOH.444, HOH.445, HOH.446, HOH.447, HOH.448, HOH.449, HOH.450, HOH.451,
HOH.452, HOH.453, HOH.454, HOH.455, HOH.456, HOH.457, HOH.458, HOH.459, HOH.460,
HOH.461, HOH.462, HOH.463, HOH.464, HOH.465, HOH.466, HOH.467, HOH.468, HOH.469,
HOH.470, HOH.471, HOH.472, HOH.473, HOH.474, HOH.475, HOH.476, HOH.477, HOH.478,
HOH.479, HOH.480, HOH.481, HOH.482, HOH.483, HOH.484, HOH.485, HOH.486, HOH.487,
HOH.488, HOH.489, HOH.490, HOH.491, HOH.492, HOH.493, HOH.494, HOH.495, HOH.496,
HOH.497, HOH.498, HOH.499, HOH.500, HOH.501, HOH.502, HOH.503, HOH.504, HOH.505,
HOH.506, HOH.507, HOH.508, HOH.509, HOH.510, HOH.511, HOH.512, HOH.513, HOH.514,
HOH.515, HOH.516, HOH.517, HOH.518, HOH.519, HOH.520, HOH.521, HOH.522, HOH.523,
HOH.524, HOH.525, HOH.526, HOH.527, HOH.528, HOH.529, HOH.530, HOH.531, HOH.532]
Variants:
- Label : Epsilon_Protonated
Component : HIS.39
- Label : Epsilon_Protonated
Component : HIS.62
- Label : Doubly_Protonated
Component : HIS.87
- Label : Epsilon_Protonated
Component : HIS.131
- Label : Epsilon_Protonated
Component : HIS.294

- Label: I
Components: [THR.1, THR.2, TYR.3, ALA.4, ASP.5, PHE.6, ILE.7, ALA.8, SER.9, GLY.10,
ARG.11, THR.12, GLY.13, ARG.14, ARG.15, ASN.16, SER.17, ILE.18, HIS.19, ASP.20,
HOH.49, HOH.54, HOH.63, HOH.70, HOH.73, HOH.74, HOH.75, HOH.76, HOH.77, HOH.78,
HOH.79, HOH.88, HOH.109, HOH.110, HOH.111, HOH.113, HOH.129, HOH.146, HOH.147,
HOH.148]
Variants:
- Label : Epsilon_Protonated
Component : HIS.19

Linear Polymers:
- { Left Terminal Component: 'A:LYS.8', Right Terminal Component: 'A:PHE.350'}
- { Left Terminal Component: 'I:THR.1', Right Terminal Component: 'I:ASP.20' }
...

The file has the following features:

  • Only the A and I entities are being simulated and so the B and J entities have been removed.
  • In entity A, the MN components have been renamed MG, ANP has become ATP, and MYR.403 has been removed as this is not relevant for the reaction.
  • In entity I, the inactive ALA.17 has been transformed into the active SER.17.
  • A number of Variant statements have been added to the A and I entities. Variants modify the composition of a component from its default state defined in pDynamo's PDB component library. In this case, all the variants specify alternative protonation states of histidine components, the default state for which is the neutral, delta-protonated form. All other components with variable protonation states take their default forms.
  • The Linear Polymer entries remain the same as in the unedited model file and specify the first and last components of the polymers in the A and I entities. It is also possible to specify the types of termination at either end of the polymer using the Left Termination and Right Termination keywords. This is unnecessary here as the default amino acid N-terminal (left) and C-terminal (right) terminations are appropriate.
  • The Link statements have been removed from the file as all links in the model are standard peptide links between amino acids and these can be handled by pDynamo's default mechanisms.

A number of additional points are important:

  • The sequence information can be changed arbitrarily in the edited model file. However, it is generally convenient to keep the sequence specification as similar as possible to the old one. This helps to facilitate comparison with the original experimental structures but it is also easier because the mapping between the old and the new sequence specifications must be given when constructing the system. This is explained in more detail in Step 3.
  • Structures in some PDB files contain duplications of part of the sequence which are often indicated by the iCode parameter of the component specification. All components found in the PDB file are automatically included in the "raw" PDB model produced in Step 1 and so the unwanted iCodes (and other components) should be removed when creating the new model file.
  • A protein (or other macromolecule) can have multiple protonation states, with significantly different properties, but the construction of a system requires that the exact atomic composition, including protonation pattern, be specified. Which one to choose?

    There are a number of approaches. One is to analyze (or visualize) the geometry around each protonation site to see if there are hydrogen-bonding or other types of interaction that favour one protonation form over another. A second is to use one of the specially-developed algorithms for calculating macromolecular pKa values. Many of these are based upon the representation of the electrostatic interactions in a system by the Poisson-Boltzmann equation but some also employ empirical criteria. Links to a number of thirdparty tools of this type with web interfaces are listed here, but future releases of pDynamo may also have something similar.

    Both approaches were used to decide on the protonation states of the histidine and other residues in the study of PKA. There was not much uncertainty in the choice in this instance, but in some cases there may be no alternative but to simulate systems with different protonation patterns.