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MAD and MIR for solving protein/DNA complexes

MAD and MIR for solving protein/DNA complexes - Protein Crystallography Forum

MAD and MIR for solving protein/DNA complexes - Discuss research on protein crystallography and crystalization. Protein Crystallography Forum.


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Old 12-25-2009, 05:46 PM
Pipette Filler
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Default MAD and MIR for solving protein/DNA complexes



Hello!

I'm a postgraduate student and just started studying X-ray crystallography. I'd like some answers about solving protein/DNA and proten/RNA stuctures, if its possible. I have read that the phasing problem can be solved either by multiple isomorphous replacement (MIR) or multiple anomalous dispersion (MAD). I have read about protein/DNA and RNA complexes that have been solved by MAD, but I haven't found any that have been solved using MIR. Are there any complexes that have been solved using MIR solely or maybe by using both MIR and MAD?

From what I understand, in order to determine the structure of a complex containing DNA or RNA, you have to substitute thymidine or uracyl with bromo-U or iodo-U or synthesize a DNA with S in place of O and then bond a Hg to the S. Are these modifications done in order to perform MAD? Can you also do these (or other) modifications in order to perform MIR? What modifications would you do to the DNA/RNA in order to perform MIR?

Probably it sounds very simple to you, but it's not to me!

Thanks!
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Old 01-12-2011, 01:11 AM
Pipette Filler
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Default Re: MAD and MIR for solving protein/DNA complexes

MAD only started coming into common usage in the mid 90s, so every protein DNA complex or DNA structure was solve by MIR before this.
The reason that bromo nucleotides and the like were never used for MIR is because they are not heavy enough. the differences for MIR you need to read are on the order of a few percent of intensity, so as to be above the measurement uncertainty. If you look at the F statistics you will see that you have to have an atom about as heavy as mercury for every 200 amino acids or so.

MIR is a hit or miss process which is why it is not used unless you absolutely have to have it. it requires that you soak the crystal in a metal salt until you get good signal. its a trial and error method that often results in the protein moving or the structure being disrupted, which of course is just plain useless for isomorphous replacement.
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complexes , mad , mir , protein or dna , solving


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