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1 Manuscript # 2004-07-21763A Supplementary Methods Additional details on the computations To determine the contribution to the total discrimination factor of 6.8 kcal/mol between oxoG and G in the active site and the alternative site (see text), additional free energy difference simulations were performed. In each case, a thermodynamic cycle was used to obtain meaningful chemical results from alchemical simulations. The first of these yielded the difference between oxoG and G in solution and in the active site; the calculated value for this difference (A1) is 4.5 kcal/mol favoring oxoG. This value was corrected for the difference in solvation free energy between oxoG and G, which was obtained by doing alchemical free energy simulations in the gas phase and in solution; the simulations yielded A2 = 4.1 kcal/mol, again favoring oxoG. Combining these two values yields 8.6 kcal/mol for the discrimination free energy in the active site and, by difference, 1.8 kcal/mol in the alternative site, both favoring oxoG, as indicated in the text. Additional details on the QM/MM free energy simulations The mixing parameter used in the alchemical free energy simulation was set equal to 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95 and 0.98. The end point singularity problem was treated by the extrapolation method1. Molecular dynamic simulations were run with a stochastic boundary potential2 for a 25 Å-radius sphere, taking N9 of the base as the center; a 1fs timestep was used. Long-range electrostatics was treated using charge-scaling procedure3. RESP charges4 for oxoG and G analogues were utilized for the Poisson-Boltzmann calculations. To simulate the free energy difference of single bases in aqueous solution, the methyl group carbon attached to N9, 2 which is shared by two alchemical species, was restrained to the center of 25 Å-radius sphere with a force constant 3 kcal/mol·Å2. Free energy derivatives were corrected using the relation of Valleau & Torrie5; the contribution is small. Convergence of the free energy simulation was controlled with the reverse cumulative averaging method5; simulations between 0.65 ns and 1.7 ns in length were required. In the reverse cumulative averaging method6, the standard errors of the energy derivatives for all the runs are calculated in the equilibrated region. The latter is determined by the normality test, with the assumption that the energy derivative data are normally distributed once the system is equilibrated. In the present study, 0.4 kcal/mol is pre-set as the upper limit of the allowed standard error of the free energy derivative, (see Eq. 6 of ref 45). Based on this criterion, simulations between 0.65 ns and 1.7 ns in length were required. The upper bound of the standard error for the overall free energy change, obtained by the integration of from 0 to 1 is on the order 0.4 kcal/mol. Data Collection and Structure Solution Data on the oxoG-complex was collected on a Rigaku R-Axis IV++ detector at Enanta Pharmaceuticals. Data on the complexes containing 7-deazaG and 7-deaza-8-azaG were collected at NSLS X4A, and processed using HKL20007. Data on the G-complex were collected at the CHESS A1 beamline and processed with DENZO and SCALEPACK7. Data collection statistics are summarized in Supplementary Table. The coordinates of the protein from the isomorphous structure of K249Q hOGG18 bound to oxoG-containing DNA was used as the initial model in refinement using CNS9. The catalytic residues and residues involved in protein-DNA interaction were omitted from the initial search model. A rigid body fit followed by energy minimization and simulated annealing performed in CNS resulted in a partial model. Electron density for the DNA and the omitted residues became clearly visible in a A- 3 weighted10 Fo-Fc map at this stage. The model was subsequently improved by iterative rounds of energy minimization, simulated annealing and grouped B factor refinement in CNS and model building in Quanta (Acelrys) while monitoring Rfree11. Simulatedannealing omit maps were frequently utilized to reduce model bias. Once the model was nearly complete, individual B-factor refinement was included. Water molecules were added to the model using both automated methods (in CNS) and manual inspection of difference maps. Amino acid side-chains of some residues were truncated at the -, -, -, or -carbon positions or the residues modelled as alanine residues if electron density was not visible for the full side-chain. Details of data collection and refinement statistics appear in Supplementary Table. 4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Simonson, T., Archontis, G. & Karplus, M. Continuum Treatment of LongRange Interactions in Free Energy Calculations. Application to Protein-Ligand Binding. Journal of Physical Chemistry B 101, 8347-8360 (1997). Brooks, C. L., III, Brunger, A. & Karplus, M. Active site dynamics in protein molecules: a stochastic boundary molecular-dynamics approach. Biopolymers 24, 843-65 (1985). Neria, E., Fischer, S. & Karplus, M. Simulation of activation free energies in molecular systems. Journal of Chemical Physics 105, 1902-1921 (1996). Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. Journal of Physical Chemistry 97, 10269-80 (1993). Berne, B. J. & Editor. Modern Theoretical Chemistry, Vol. 5, Pt. A: Statistical Mechanics: Equilibrium Techniques (1977). Yang, W., Bitetti-Putzer, R. & Karplus, M. Chaperoned alchemical free energy simulations: A general method for QM, MM, and QM/MM potentials. Journal of Chemical Physics 120, 9450-9453 (2004). Otwinowski, Z. & W., M. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326 (1997). Bruner, S. D., Norman, D. P. & Verdine, G. L. Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature 403, 85966 (2000). Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905-21 (1998). Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 104-149 (1986). Brunger, A. Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr. D Biol Crystallogr 49, 24-36 (1993).