Hi James,
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.
Cheers,
Paul
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:
> http://bl831.als.lbl.gov/~jamesh/movies/index.html#reso
>
> 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.
>>
>> Cheers,
>>
>> Ed.
>>
--
Paul Adams
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Division Deputy for Biosciences, Advanced Light Source, Lawrence Berkeley Lab
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