[phenixbb] FW: phenixbb Digest, Vol 87, Issue 3
Phoebe A. Rice
price at uchicago.edu
Sun Feb 10 13:52:33 PST 2013
Although its theoretically possible, I've never had 5-Br-dU cause a problem. My approach would be to pick the strand with more T's and get every T replaced with 5-Br-dU, unless the 3' nt is a T (you pay a lot more for 3' modifications).
We have had issues where crystals wouldn't grow with a heavily 5-I-dU modified DNA, presumably because Iodine is significantly bigger than a the 5-methyl of T, and it annoys the protein. If you've got good footprinting data, you may be able to guess where the protein will "care" about the surface of the major groove, and avoid putting iodines in those regions. If not, its probably faster to just order several oligos and try them all. If you've got a lot of T's and are worried about locating all those heavy atoms, I'd go for one derivative with a couple of paired 5-I-dU's (in spots where you have two T's in a row) in hopes that that will give you a toehold on solving the Patterson, and then a 2nd where every T on one strand is brominated, which may provide more phasing power.
Note that both of these pop off in the x-ray beam, so collect your anomalous data first and fast. Also note that if you're lucky, you can get an extra large delta f' by subtracting the last data set from the first ("radiation induced phasing").
Hope that helps!
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; PRice at uchicago.edu
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