Wow, I got quite a number of responses. Thanks everyone. Let's elaborate.
Petr, I don't know my anomalous signal, because I haven't yet done the
experiment. If I had, I would have definitely talked about chi2 values
for I+/I- merged and unmerged, Anomalous Patterson maps, or other
measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great
paper to read on that, I suggest every grad student to present it in
their Crystallography Journal Club).
I was merely pondering today, but I plan to do the experiment very soon
(crystals are waiting for synchrotron time).
About your example, 2.9 A diffracting crystals (with 4 A anomalous
signal) are in the doable range, as you suggested. The question is what
would happen if your crystals diffract to 4 A, and anomalous signal dies
at 6 A. The interesting bit of course is 1 Met per 200 residue, which
should put to death the "1 in 50" or "1 in 100" Methionine myths: it
depends on the quality of your data.
Engin
On 5/10/09 2:50 PM, Leiman Petr wrote:
Dear Engin Ozkan,
You have told us how bad your crystals are, but you did not mention how good
your anomalous signal is:
1. To what resolution does your anomalous signal extend and what statistic is
used for this estimate?
2. Do your dispersive and Bijvoet Pattersons look similar and what is the
measure of similarity?
This structure
http://www.pdb.org/pdb/explore/explore.do?structureId=1K28
which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire
complex),
was solved using a chimerical SeMet derivative, in which one protein was SeMet
labeled (17 Se per a.u.) and the other was native.
The Semet dataset had a detectable anomalous signal to 4 A resolution (at
most). The diffraction extended to 2.9A resolution.
Sincerely,
Petr
-----------------------------------------------------------
Petr Leiman
Institut de physique des systèmes biologiques
École Polytechnique Fédérale de Lausanne (EPFL)
Cubotron/BSP-415
CH-1015 Lausanne
Switzerland
-----Original Message-----
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of
Engin Ozkan
Sent: Sunday, May 10, 2009 11:01 PM
To: [email protected]
Subject: [ccp4bb] phasing with se-met at low resolution
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.
