a recent experience in our lab with molecular replacement (wt and
disordered point mutant; same space group and unit cell)
was solved with a combination of two methods.
1. We made omit maps in the disordered region at several lower
resolutions. The region became interpretable after suffereing through
these maps, building residue by residue and refinement.
2. Then we had the bright idea to make Fwt-Fmutant maps to confirm
our interpretation. Happily this map did confirm the unexpected large
structural changed caused by a point mutant.
On Fri, Apr 13, 2012 at 1:31 PM, James Holton <[log in to unmask]> wrote:
> Francis,
>
> I think in the cases you describe the region in question is disordered.
> Time and time again I have users coming to my beamline wanting to clean up
> a "questionable region" by getting experimental phases. Ahh! If only I had
> a nickle for each one. Oh wait, I suppose I kind of do? I take that back!
> Go MAD everyone!
>
> Much as I hate to discourage people from using my favorite technique, Tim is
> right: phases are not region-specific in electron density maps. Dale does
> make a good point that there is such a thing as "model bias" and one can
> argue that experimental phases don't have it. But, this is only true if you
> have not yet applied solvent flattening. How long has it been since you
> looked at a "raw" experimentally-phased map (before solvent flattening)?
> I'm willing to bet a while. With very few exceptions, raw experimental
> phases are lousy. We have actually become quite dependent on density
> modification to clean them up. In fact, solvent flattening is the only
> reason why SAD works at all.
>
> However, you CAN use anomalous differences to clear up disordered regions in
> a different way. Something I started calling "SeMet scanning" a number of
> years ago. A few of my users have done this, and a good example of it is
> Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate
> residues in the disordered region one at a time to SeMet, and look at phased
> anomalous difference Fourier (PADF) maps. These maps are surprisingly
> clear, even when the anomalous difference signal is so weak as to make
> experimental phasing hopeless. Yes, the best phases to use for PADF maps
> are model phases, but, as always, it is prudent to refine the model after
> omitting the thing you are looking for before calculating such phases.
>
> Another way to get residue-specific labeling for low-resolution chain
> tracing is radiation damage. If you expose for the right amount of time,
> Asp and Glu side chains will be specifically "burnt off", but not Asn and
> Gln. You will also see Met loosing its head, etc. So, as long as you have
> read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to
> guide sequence assignment, even at 4.5 A and worse.
>
> Anyway, when it comes to the question of "is it disordered or is it model
> bias?", I think it is usually the former. It is very difficult to make
> "model bias" suppress a region that is actually well-ordered. Try it!
> After all, this is the whole reason why we bother looking at fo-fc maps.
> Then again, it is always possible to have a model so bad that the phase
> error is enough to squash anything. An excellent example of this can be
> found in the Book of Fourier. Taking amplitudes from the image of a cat,
> you can see what happens when you use the phases of a duck:
> http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif
> as opposed to what happens if you use the phases of a manx:
> http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif
> A manx is a species of cat that doesn't have a tail, so no animals were
> harmed in obtaining these phases. My point here is that the cat's tail can
> be seen quite readily in the 2fo-fc map if most of the structure is already
> "right", but if your model is completely unrelated to the true structure
> (fitting a duck into a cat-shaped hole), then everything is "in the noise".
>
> Real structures are usually somewhere between these two extremes, and I
> think an important shortcoming in modern crystallography is that we don't
> have a good quantitative description of this middle-ground. We all like to
> think we know what "model bias" is, but we don't exactly have "units" for
> it. Should we be using a scale of 0 to 1? Or perhaps "duck" to "cat"?
> Yes, I know we have "figure of merit", but FOM is not region-specific.
>
> In my experience, as long as you have ~50% of the electrons in the "right"
> place (and none of them in the "wrong" place), then you can generally trust
> that the biggest difference feature in the fo-fc map is "real", and build
> from there. As the model becomes more complete, the phases should continue
> to get better, not worse. Eventually, this does break down, although I'm
> not really sure why. With small molecules, the maximum fo-fc peak keeps
> getting bigger (on a sigma scale) as you add more and more atoms, and the
> biggest one you will ever see is the last one. For macromolecules, the
> difference features keep getting smaller and smaller as you build. Perhaps
> small errors (like non-Gaussian atomic displacement distributions being
> modeled as Gaussians) slowly accumulate? Perhaps there are other sources of
> systematic error that we don't yet fully understand? Eventually, for
> whatever reason, you stop building. Having electrons in the wrong place is
> about twice as bad as not having them at all, which I think is why we trim
> models so aggressively for molecular replacement, and also why we are so
> reticent to model in things that we are not "sure" about. Disordered
> regions, of course, will always lie at a lower level of electrons/A^3 than
> ordered regions, and therefore will be the last things to show up as the
> "top peak" in the fo-fc map. They will also be the last things to poke
> their heads above the 1-sigma contour in a 2fo-fc map, but that does not
> mean they are "not there". You can lower the map contour and see them
> easily enough (even in 3bcl). The trick is having some kind of
> statistically-sound rule for "being sure". Otherwise, we might start seeing
> "map contour creep", just as we currently see "R factor creep" in
> high-profile journals.
>
> -James Holton
> MAD Scientist
>
>
> On 4/10/2012 1:04 PM, Francis E Reyes wrote:
>>
>> Dale
>>
>> Thank you for the case study. I will certainly remember it when I next
>> see:
>>
>>> "I don't see density for these atoms therefore they must be
>>> disordered."
>>
>> You do mention though, that when you were able to assign the sequence to
>> the beta sheets, that the loop regions became clear.
>>
>> I consider the case (which a majority of cases seem to be), where the
>> author has built and sequence assigned 95% of the ASU, but is unable to
>> model a loop region. One possibility is that the loop is truly disordered
>> (95% of the ASU is built and is presumably right), the other possibility is
>> that there's an inherent error in the existing structure that is affecting
>> the interpretation of the loop region. The errors are probably extremely
>> subtle and distributed throughout the model (think of the improvements DEN
>> refinement gave for the rerefinement of p97).
>>
>> I guess in either case, because of the dependency of the map on the
>> existing set of phases it's difficult to determine whether it's truly
>> disordered or not.
>>
>>
>>> P.S.: The author should not look at an 2fofc-map but a
>>> sigma-A-weighted map to reduce model bias.
>>
>> Tim,
>>
>> I assume a sigmaA weighted 2Fo-Fc map (which I believe is the default for
>> most crystallographic refinement packages).
>>
>> F
>>
>> ---------------------------------------------
>> Francis E. Reyes M.Sc.
>> 215 UCB
>> University of Colorado at Boulder
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