Lab notebook for Enzyme Binding and Specificity (download 13Mb)(Back)

This notebook details all the steps involved in the calculation of the binding energy and specificity energy of 8-Oxo-dGMP in the nucleotide-pool sanitizing enzyme MTH1, starting with the un-modified PDB file, and ending with the individual energies.
Excel page 1
 Starting Model		  
Steps used in the original journal article (Uses the old "ADD-H" method)  
Step Operation   Hyperlinks ΔHf  
Step 4 Optimize entire system using bias=3 Arc file JSmol -24432.664  
Step 5 Optimize atoms within 5Å of 8OG
Data-set
1SCF Output Arc file JSmol -24446.180  
 
 
Steps used using the new "ADD-H" hydrogenation method  
Step Operation   Hyperlinks       ΔHf  
Step 1 Isolate MTH1 Chain "A" Data-set Output Arc file JSmol -  
Step 2 Add hydrogen atoms Data-set 1SCF Output Arc file JSmol -20775.780  
Step 3 Optimize Hydrogen atoms' positions Data-set 1SCF Output Arc file JSmol -23049.071  
Step 4 Optimize entire system using bias=3 Data-set 1SCF Output Arc file JSmol -24491.919  
Step 5 Optimize atoms within 5Å of 8OG Data-set 1SCF Output Arc file JSmol -24500.463  
 
Compare pairs of geometries  
Geometries Data-set JSmol Output  
Old 4 and old 5 Data-set JSmol Output  
New 5 and old 5 Data-set JSmol Output  
 
New 4 and new 5
Data-set
JSmol
Output
 

In the original journal article, "A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1" an older version of the utility ADD-H was used. In the newer version of ADD-H the positions of hydrogen atoms in water molecules are pre-optimized using a very primitive geometry optimizer. This produces a better starting geometry.  For completeness, the geometries in steps 4 and 5 of the preparation of the starting model are given at the start of "Excel page 1: Starting Model". 

This is followed by the complete procedure for generating the starting model using the newer ADD-H utility, starting with the un-modified PDB file for 3ZR0 and ending with the 8-Oxo-dGMP system that can be using in the prediction of the binding and specificity.

The effect of removing the effects of the bias on the atoms near to the substrate can be seen by the comparison of the old 4 and old 5 geometries.  Large changes occur in water molecules near the phosphate group, but as these are far from the binding site, the influence of these charges on binding and specificity is unimportant.

Both starting models, the one using the older ADD-H and the one using the newer ADD-H, should give similar results.  The geometry of the important part of the system, i.e., the region where binding occurs, is essentially the same in both cases, as can be seen in a comparison of the old and new starting models.







Excel Page 2    
  Raw data used in calculating Stabilization and Specificity        
Chain "A" Chain "B"    
Residue NUL 8OG GMP 8OG   GMP    
             
     Asp 119 -23644.89 -24328.98 -24262.89 -19307.02   -19240.03    
     Asp 120 -23655.83 -24330.09 -24263.39 -19298.64   -19231.53    
     Asn 33 -23773.55 -24365.64 -24300.19 -19351.28   -19231.53    
     Leu 9 -23814.97 -24415.26 -24349.12 -19392.43   -19325.05    
     Lys 23 -23893.81 -24492.08 -24426.02 -19471.20   -19404.28    
     Met 81 -23828.00 -24434.66 -24369.51 -19408.82   -19404.28    
     Trp 117 -23847.75 -24449.51 -24385.22 -19436.32   -19371.43    
     Phe 27 -23854.25 -24457.48 -24392.03 -19434.34   -19366.94    
     Val 83 -23821.83 -24425.61 -24359.06 -19400.40   -19333.19    
     Phe 72 -23847.93 -24449.37 -24385.14 -19434.34   -19360.73    
     Water 2134 -23765.49 -24366.33 -24301.07 -19342.02   -19275.73    
     Trp 123 -23845.34 -24450.29 -24382.71 -19424.93   -19357.45    
     Phe 139 -23842.76 -24448.59 -24383.16 -19424.55   -19358.74    
     
No mutations -23840.26 -24446.41 -24380.00 -19421.98   -19354.70    
No mutations - for 119 and 120 only -23756.25            
12 Mutations -23436.03 -24097.97 -24118.32 -19173.24   -19114.79    

Page 2 holds a reconstruction of the raw data used in the journal article.  Because this is a reconstruction, there are minor differences, typically 0.001 kcal·mol-1, in heats of formation.

Also, because of the improved ADD-H function, the results in Page 2 can no longer be reproduced de novo.  However, the individual files used in generating the entries in the tables are available in the ZIP file, and if these are run, the results reported in the paper can be reproduced.

Also, the results in Page 1 can be used as the starting point for reproducing page 2, although this would be a lot of work!

 

 

 

 

 

 

 

 

 

        Excel Page 3            
         Stabilization and Specificity          
Residue Diff. 8OG-NUL   Stabilization Diff. GMP-NUL Stabilization Specificity    
     
Asp 119 -684.09   -6.07 -618.00 -5.74 -0.32    
Asp 120 -674.26   -15.89 -607.56 -16.19 0.29    
Asn 33 -592.09   -14.06 -526.64 -13.11 -0.96    
Leu 9 -600.30   -5.85 -534.15 -5.59 -0.26    
Lys 23 -598.27   -7.88 -532.21 -7.53 -0.35    
Met 81 -606.66   0.51 -541.51 1.77 -1.26    
Trp 117 -601.76   -4.39 -537.47 -2.28 -2.12    
Phe 27 -603.23   -2.92 -537.78 -1.96 -0.96    
Val 83 -603.78   -2.37 -537.23 -2.51 0.14    
Phe 72 -601.43   -4.72 -537.20 -2.54 -2.18    
Water 2134 -600.84   -5.31 -535.58 -4.16 -1.14    
Trp 123 -604.95   -1.20 -537.37 -2.37 1.17    
Phe 139 -605.83   -0.32 -540.40 0.66 -0.98    
Sum:   -70.47 Sum: -61.55 -8.92    
No mutations -606.15   -539.74    
No mutations - for 119 and 120 only -690.15   -623.75    

This page uses the Excel arithmetic options.  So if a change is made to Page 2, Page 3 is automatically updated.  That is, if an error is detected in a raw-data table, when the error is removed, all array elements that depend on that entry are immediately and automatically updated.