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Arbitrary Barrier cutoff

Was there any other reason you chose this arbitrary barrier cutoff besides to show agreement with your computational approach? How should people relate this cutoff to studies of other proteins? I can't seem to find a plot of experimental vs. computationally determined barrier heights, which would be very valuable to a reader, I am just missing it? If not could you please post one? Thanks.

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Accepted answer

"Was there any other reason you chose this arbitrary barrier cutoff besides to show agreement with your computational approach?"

No. As we write two paragraphs further down: "We note that defining the cutoff is done purely for a post hoc comparison of experimental and computed data. When using the computed barriers to identify promising experimental mutants, one simply chooses the N mutants with the lowest barriers, where N is the number of mutants affordable to do experimentally (e.g., 20 in the discussion of set L)"

"I can't seem to find a plot of experimental vs. computationally determined barrier heights, which would be very valuable to a reader, I am just missing it?"

As we write in the preceding paragraph: "Given the approximations introduced to make the method sufficiently efficient, it is noted that the intent of the method is not a quantitative ranking of the reaction barriers, but to identify promising mutants for, and to eliminate non-promising mutants from, experimental consideration. Therefore only qualitative changes in overall activity are considered, which are represented by the activity factors ( + 1/−1)"

Furthermore, the experimental activity assay data reflects more than kcatand can not be directly, quantitatively linked to a barrier.

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Accepted answer

Dear Josiah

Thanks for your question, which also has been raised previously.

In this study we have a relatively big set of experimental data points we can compare the computational data against. So with the cutoff we can estimate the highest possible accuracy of the method, given all the approximations. From this we can get an idea on how reliable predictions will be for mutants of this enzyme where no experimental data is available.

"How should people relate this to studies of other proteins?"

This point only becomes relevant when you have a relatively large number of experimental data points available, which is rarely the case.

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