Mutations to 3-methyladenine

Recently, a 3DNA user asked on the Forum about how to perform mutations to 3-methyladenine. The user reported that the procedure described in the FAQ entry How can I mutate cytosine to 5-methylcytosine did not work for the case of 3-methyladenine. This ‘limitation’ is easily understandable: the 3DNA mutate_bases program must have knowledge of the target base, 3-methyladenine, to perform the mutation properly. The program works for the most common 5-methylcytosine mutations since the corresponding 5MC file (Atomic_5MC.pdb, in the standard base-reference frame) is already included within the 3DNA distribution. By supplying a similar file for the target base, mutate_bases runs the same for mutations to 5-methylcytosine (or other bases). This blog post outlines the procedure, using 3-methyladenine as an example.

A ligand name search for 5-methylcytosine on the RCSB PDB led to only two matched entries: 2X6F and 3MAG. The ligand id is 3MA. Since 3MAG has a better resolution (1.8 Å) than 2X6F (3.3 Å), its 3MA ligand was extracted from the corresponding PDB file (3MAG.pdb). The atomic coordinates, excluding those for the two hydrogens, are as below. Note that the 3-methyl carbon atom is named CN3.

HETATM 2960  N9  3MA A 600      16.587  14.258  22.170  1.00 49.87           N
HETATM 2961  C4  3MA A 600      17.123  13.100  21.622  1.00 50.46           C
HETATM 2962  N3  3MA A 600      16.877  11.811  22.009  1.00 50.37           N
HETATM 2963  CN3 3MA A 600      15.983  11.363  23.063  1.00 50.41           C
HETATM 2964  C2  3MA A 600      17.590  10.968  21.241  1.00 50.11           C
HETATM 2965  N1  3MA A 600      18.422  11.217  20.224  1.00 49.27           N
HETATM 2966  C6  3MA A 600      18.627  12.484  19.858  1.00 48.99           C
HETATM 2967  N6  3MA A 600      19.426  12.709  18.829  1.00 46.12           N
HETATM 2968  C5  3MA A 600      17.949  13.503  20.593  1.00 49.89           C
HETATM 2969  N7  3MA A 600      17.929  14.900  20.488  1.00 49.84           N
HETATM 2970  C8  3MA A 600      17.113  15.286  21.434  1.00 49.58           C

After running the 3DNA utility program std_base with options -fit -A, the corresponding atomic coordinates of 3MA are transformed to the standard base reference frame of adenine. The file must be named Atomic_3MA.pdb, and it has the following contents:

HETATM    1  N9  3MA A   1      -1.287   4.521   0.006  1.00 49.87           N
HETATM    2  C4  3MA A   1      -1.262   3.133   0.004  1.00 50.46           C
HETATM    3  N3  3MA A   1      -2.337   2.286  -0.009  1.00 50.37           N
HETATM    4  CN3 3MA A   1      -3.743   2.648  -0.047  1.00 50.41           C
HETATM    5  C2  3MA A   1      -1.905   1.013   0.001  1.00 50.11           C
HETATM    6  N1  3MA A   1      -0.662   0.520   0.004  1.00 49.27           N
HETATM    7  C6  3MA A   1       0.366   1.372  -0.003  1.00 48.99           C
HETATM    8  N6  3MA A   1       1.588   0.867  -0.034  1.00 46.12           N
HETATM    9  C5  3MA A   1       0.068   2.768   0.003  1.00 49.89           C
HETATM   10  N7  3MA A   1       0.875   3.914  -0.003  1.00 49.84           N
HETATM   11  C8  3MA A   1       0.026   4.909  -0.003  1.00 49.58           C

Note that in file Atomic_3MA.pdb, (1) the z-coordinates of the base atoms are close to zeros, (2) the ordering of atoms is as in the original ligand of 3MA shown above.

With Atomic_3MA.pdb in place (in the current working directory, or the $X3DNA/config folder), one can perform 3-methyladenine mutations using mutate_bases. For illustration purpose, let’s generate a B-form DNA with base sequence GACATGATTGCC using the 3DNA fiber program:

fiber -seq=GACATGATTGCC fiber-BDNA.pdb

To mutate A7 to 3MA, one needs to run mutate_bases as following:

mutate_bases "chain=A s=7 m=3MA" fiber-BDNA.pdb fiber-BDNA-A7to3MA.pdb

The result of the mutation is shown in the figure below. Note that the backbone has identical geometry as that before the mutation, and the mutated 3MA-T pair has exactly the same parameters (propeller/buckle etc) as the original A-T. These are the two defining features of the 3DNA mutate_bases program.

3DNA 3-methyladenine mutation

Please see the thread mutations to 3-methyladenine on the 3DNA Forum to download files fiber-BDNA.pdb and fiber-BDNA-A7to3MA.pdb.

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