Dear James,
I don't understand why measuring anomalous differences has nothing to do with
resolution.
Heavy atoms
scatter anomalously because the inner shell electrons
of the heavy atom cannot be considered to be free anymore
as was assumed for normal Thomson scattering. As a result
the atomic scattering factor of the heavy atom becomes
complex and this compex contribution to the structure
factor leads to non-equality of Friedel pairs in non-centro
symmetric systems(excluding centric zone). This feature is taken advantage in
phase determination. Since the inner shell electrons
being relatively more strongly bound in heavy atoms
contribute to anomalous scattering and its effect
is more discernable for high angle reflections . Here
the anomalous component of the scattering do not
decrease much because of the effectively small atomic
radii (only inner shell being effective). FOR HIGH
ANGLE REFLECTIONS ANOMALOUS DATABECOMES IMPORTANT.
Raja
________________________________
From: James Holton <[email protected]>
To: [email protected]
Sent: Tuesday, 12 May, 2009 11:26:55 AM
Subject: Re: [ccp4bb] phasing with se-met at low resolution
measuring anomalous differences has nothing to do with resolution.
measuring anomalous differences has nothing to do with Rmerge.
measuring anomalous differences has EVERYTHING to do with signal and noise.
(as does measuring anything else)
If your average anomalous difference is going to be ~5%, then you need to be
able to measure a 5% change in spot intensity, yes? So, if you take your
native data, and compare the merged values of I+ and I- (known in Scala as
Ranom), and they are already more than 5% different, then ... you are in
trouble. But if Ranom for native data is less than 5%, then you stand a chance
of measuring a 5% difference.
That is, for native data, the "true" values of I+ and I- should be "the same"
(within the Bijvoet ratio for the sulfurs, which is usually < 0.5%), so
comparing I+ and I- for native data is actually a very good way to get your
expected "anomalous error". You can improve this number by increasing
redundancy, even if you reduce the exposure time to compensate. In fact, it is
a VERY good idea to do this when trying to measure anomalous differences.
Redundancy is good for anomalous, but bad for high-res data. Long exposures
and fine slicing are good for high-res data, but bad for anomalous.
Resolution comes into play because the "anomalous error" will approach infinity
as your spot intensity approaches zero, so you will never be able to measure
anomalous differences for your highest resolution bin. The resolution to which
you CAN measure anomalous differences (with a signal-to-noise ratio greater
than one) will be the resolution where the cumulative Ranom rises to the
Bijvoet ratio (5% in your case). That is, look for the resolution limit where
the overall "native Ranom" is 5%, and that is the resolution to which you will
probably get experimental phases.
If there is no such resolution limit (Ranom > 5% in all bins), then MAD/SAD
will not work with your current data collection method. Higher redundancy is
called for.
However, do not get too excited if this resolution limit is 6 A. Although 6 A
phases are better than no phases at all, have you ever LOOKED at a 6 A map? It
can be very hard to tell if it is protein or not, even with perfect phases and
all the right hand choices, etc. Programs and crystallographers alike can get
confused by this. I know that there are still many structural biologists out
there who "just want to get the structure", but I remind you that you can
already "get the structure" to ~50 A resolution with other techniques. Such as
gel filtration.
The success of phase extension does depend on resolution. Although I do not
have a quantitative argument for it, the success of SAD structure determination
at worse than 4 A does seem to drop precipitously. This could simply be
correlated with the crappiness of the crystals, but it is important to remember
that SAD relies heavily on density modification technology, such as solvent
flattening and histogram matching, etc, and these methods loose a great deal of
power as the resolution of the map decreases (and the protein-solvent contrast
becomes less clear). IMHO it is ALWAYS better to collect MAD data, because
then the dichotomous phase ambiguity is resolved experimentally. Two
wavelengths are twice as good as one, even with the exposure time cut in half.
-James Holton
MAD Scientist
Engin Ozkan wrote:
> Hi everyone,
>
> I thought I start a new thread while it is unusually quiet on the bb. I am
> pondering over the practical limitations to MAD and SAD phasing with Se-Met
> at low resolution. What is the lowest resolution at which people have solved
> structures "only" using phases from selenium in a "realistic" case? Let me
> further qualify my question: My *realistic* *low* resolution case is where
> 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy).
> 2. Resolution limit is worse than 3.5 Angstroms, where <I>/<sigma> in the
> last resolution bin is between 1 and 3 (i.e. your crystals are really lousy).
> 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression
> systems, so 100% is not usually achieavable),
> 4. The number of selenium atoms are enough many that the Crick-Magdoff
> equation would give you *at least* an average 5% change in intensities
> (assuming 6 electrons contributed per selenium, based on both absorptive and
> dispersive differences being at about 6 e- at the absorption edge).
> 5. and specifically, no other phases and molecular replacement solutions are
> available.
>
> Obviously, I have a case very similar to what's described above, and three
> years of failure with heavy atom derivatization (I am still trying). I would
> be happy to hear about Se-Met cases, and data collection strategies (2wl vs.
> 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references
> of them. Again, no other partial phases, and no data cut off at 3.6 A with an
> I/s of 15 in the last resolution bin. Are there any examples out there?
> Searching the RCSB and PubMed did not point out to me many successful cases.
>
> Thanks,
>
> Engin
>
> P.S. I would also appreciate the specific query type for searching the PDB on
> the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have
> everything under the sun searchable, but I cannot find this one.
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