my understanding is that phenix.refine allows any number of alternate conformers. There may have been a limit of 4 some time in the past, but no longer. So your idea could be tested.
On Mar 27, 2012, at 12:33 PM, James Holton wrote:
> Try this:
> 1) take your favorite PDB file and set all the B factors to ~80 (reduces series-termination errors)
> 2) use sfall/fft in CCP4 to calculate structure factors to 4A resolution
> 3) use sftools to add a "SIGF" column (0.1 will do) to make refmac5 happy
> 4) refine the "perfect" model against these fake data for ~5 cycles (with "solvent no")
> 5) load this up in coot and contour at 1 sigma
> 6) repeat the refinement with a PDB file containing only main chain.
> 7) repeat the refinement after putting all the side chains in their most likely (Ponder-Richards) rotamers.
> Ask yourself these questions:
> 1) can you "see" the side chains?
> 2) can you "see" the waters?
> 3) what are the R factors from these refinements?
> Answers: 1) no, 2) no, 3) ~3% for "perfect", ~50% for "main chain", and ~36% for "likely rotamer"
> Now ask yourself: even though there is "no density" for side chains and waters, is there really "no evidence" that they exist?
> The point I am trying to make here is that you EXPECT side chains to poke out of density at low resolution, even under ideal conditions (perfect phases). For example, the C-deltas of Leu will "breach" the 1-sigma contour at around 2.8A resolution and worse. You can see this in my old movie:
> When it comes to building, yes, once an atom dips below the 1-sigma contour it gets harder and harder to know exactly where it is, but it does have to be somewhere. Somewhere nearby. Formally, there is "prior knowledge" of bond lengths, etc. at play. And if you know that there is one copy of a given atom in every unit cell of the crystal, then occupancy < 1 is inappropriate. Much better to use B = 999, which models the atom as a Gaussian with the electrons spread over an area about 3.5 A wide. This is roughly the range your average side chain atom has available to it, given that it is attached to the main chain by covalent bonds.
> Of course, a more "Bayesian" model for the "I don't know what the rotamer is" situation would be to build in ALL possible rotamers, with occupancies equal to their Ponder-Richards probabilities. Some improvement to this initial "guess" would no doubt be made by using constrained occupancy refinement of rigid-body side chains. Unfortunately, this is impossible with any refinement program I know about, since refmac, phenix.refine, etc. don't support more than 3 or 4 alternate conformers.
> Building in all possible conformers and using the occupancy as a "p-value" would also help solve the problem of the careless and/or uneducated over-interpreting PDB files. Which is the "right one"? Good question! I think its time we started dispelling the myth of the single-conformer protein anyway.
> -James Holton
> MAD Scientist
> On 3/26/2012 7:40 AM, Ed Pozharski wrote:
>> On Mon, 2012-03-26 at 10:17 -0400, Gregory Bowman wrote:
>>> But what about the issue of resolution? As was previously pointed out,
>>> at say 3.2 Å resolution, many side chains will fail to fit, but it
>>> doesn't seem appropriate to trim them all down.
>> Why is it inappropriate to trim them down? Sometimes at low resolution
>> all one can be confident about is the backbone trace.
>> Just to be clear, I am talking about atoms whose positions are not
>> supported by electron density, i.e. where difference map in the absence
>> of the side chain is featureless. I assume that is the likely situation
>> when one would set occupancy to zero.
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