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.
