Dear Chen,
If you don't know how many sites to expect - and in some cases, e.g. an
iodide soak, this is inevitable - I recommend trying different numbers
(FIND N in shelxd) and choosing the one where the occupancy of peak N
comes out as about 0.2. If it is higher you need a higher N and vice
versa. hkl2map makes a nice plot of the occupancies but you can also
simply look at the values in the .res file. You have to be careful with
sites on special positions because the occupancy is divided by the
multiplicity of the position. Except for some soaks, it is unlikely that
there will be an atom on a special position so it is recommended that
the second MIND parameter is set to say 2.2 (the default) to reject
peaks on special positions. In borderline cases you will also have to
try different resolutions for truncating the anomalous data (e.g. SHEL
99 2.0), but if you are looking for disulfide sulfurs you may also need
to search for M disulfides in the peaksearch (DSUL M and MIND 3.5)
because if the resolution cut is higher than 2A the individual sulfurs
may not be resolved.
The current shelxe allows you to refine the heavy atoms before doing
density modification (-zN for a maximum of N sites. for SAD you should
also specify -h unless you are using a separate higher resolution native
dataset). This should always be used except when you had to use DSUL in
shelxd, because shelxe does not (yet) know about disulfides.
Heavy atom Pattersons are inevitably noisy, especially for weak SAD
data, both because of the low I/sigma of the anomalous differences and
because we are using the anomalous difference as an approximation to the
true heavy atom structure factor. Pattersons calculated from MAD FA
values do not suffer from this approximation but may be affected more by
radiation damage. The Patterson is still useful to see if any heavy
atoms are present, but the above trial and error method is a better way
of determining the number of heavy atoms. This also explains why some
Patterson density may be negative, especially close to stronger positive
peaks, other reasons in the case of SAD are the missing reflections in
centrosymmetric projections and possible incomplete coverage of Friedel
pairs.
Best wishes, George
On 04/02/2014 04:16 AM, Chen Zhao wrote:
By the way, I have another question related to the number of sites. I
just rethought about what Herman mentioned, and I just realized that
the number of sites, at least strong sites, could be guessed from an
anomalous Patterson map. Therefore I looked at the anomalous Patterson
of my data. The map indicates that the signal is very weak, and there
are only 4 non-origin positive peaks (3 effective peaks, because 2 are
symmetry equivalent), indicating there are at most 3 heavy atoms
(3x2=6) if all of them contribute to the map. However, in shelxd, I
can get optimal results from 6 sites out of the same data set. I am
not sure whether this is because the anomalous Patterson is not
sensitive enough. Now I am more confused about the criteria in
choosing the parameters when running shelx and all other programs.
What should be the general practice? Should I rely more on the
anomalous Patterson or more on the better scores reported? When I
tried to optimize the results by changing the parameters (either when
locating sites or when phasing), am I more heading for the real signal
or more simply tweaking the parameters? Maybe the golden standard
would be whether you could solve the structure.
Another question is, although from the Euler's equation I could
understand negative Patterson peaks (maybe I am wrong), I am still not
clear what negative Patterson peaks represent and why people generally
don't talk about it. In the specific data I am talking about above,
there is a negative Patterson peak at the same position as a positive
Patterson peak with the same height. Is there any indication about this?
Thank you so much for your attention!
On Tue, Apr 1, 2014 at 2:00 PM, Nazia Nasir
Phd2009,ProteinCrystall.Lab <[email protected]
<mailto:[email protected]>> wrote:
Dear Jurgen,
The beam position is fine. we have collected many data sets before
and after this data. Moreover, we the Technical scientist always
checks the beam position before we mount the crystals.
Thanks
On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch <[email protected]
<mailto:[email protected]>> wrote:
check your beam position
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742 <tel:%2B1-410-614-4742>
Lab: +1-410-614-4894 <tel:%2B1-410-614-4894>
Fax: +1-410-955-2926 <tel:%2B1-410-955-2926>
http://lupo.jhsph.edu
On Apr 1, 2014, at 1:26 PM, Nazia Nasir
Phd2009,ProteinCrystall.Lab <[email protected]
<mailto:[email protected]>> wrote:
Dear all,
I am just taking advantage of this particular thread to add
my query also. I hope you don't mind Chen.
We haven't solved any structure in our lab using SAD phasing,
so pardon me for sounding naive.
I have a 6.5 A data of anomalous scattering with a 3.5 A data
using the Cu anode.My crystals dont grow any better. I have
around 10 Met distributed fairly evenly through out the
sequence. So is it possible to get any anomalous signals form
this data?
Moreover, I am not able to index my 3.5A data at all. The
spots don't fit well. What could be the problem?
Hope this thread can be of benefit for both me and Chen.
Thanks
On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao <[email protected]
<mailto:[email protected]>> wrote:
Dear Herman,
Thank you so much for your suggestions. The density that
passes through the rotational axis is so strong and
extended that can be considered as a significant portion
of the molecule. However, some density in the middle
might show some features. I have no experience and this
could be only artifact.
The unit cell seems to be able to fit 1-2 molecules. But
if there is one molecule/ASU, the solvent content is
about 89%, which is possible but unlikely. Although the
resolution of the crystal is not high, it is rather rigid
and easy to handle, which might indicate a not-too-high
solvent content. I soaked the native crystal with heavy
atom compounds and I have no clear idea of the
relationship between metal binding and sequence, so I
don't know how many sites to expect.
Best,
Chen
On Tue, Apr 1, 2014 at 9:42 AM,
<[email protected]
<mailto:[email protected]>> wrote:
Dear Chen,
I am not an expert on SAD and MAD. However, at this
stage I would not worry too much about density going
through the 2-fold axis. There might be a sulfate ion
or some other buffer component present at that
position, or it may just be an artifact that will go
away once the structure has been built and refined.
The questions I would worry about is: how much too
small is your unit cell? Is it just crowded, say
25-30% solvent, or would your protein molecule not
fit at all? Does the amount of solvent as estimated
from your SAD/MAD maps agree with the amount of
solvent obtained from the calculation of the Matthews
volume? How many (SeMet?) sites do you expect, 6,
more, less? If everything looks ok except that the
unit cell is rather crowded, I would go ahead and try
to build the structure. __
However, if even a single protein molecule would not
fit in your unit cell, or you find many more sites
than you can explain, you should start worrying about
twinning. Even than the structure can probably be
solved, but then you need some real experts!
My 2 cents,
Herman
*Von:*CCP4 bulletin board
[mailto:[email protected]
<mailto:[email protected]>] *Im Auftrag von *Chen
Zhao
*Gesendet:* Montag, 31. März 2014 23:46
*An:* [email protected]
<mailto:[email protected]>
*Betreff:* [ccp4bb] Space group problem?
Dear all,
I am now trying to phase a structure in C2 using
anomalous scattering at 5-6 A. It is hard to improve
the derivative resolution at the moment. Shelxd is
able to locate 6 sites with a distinct CC and FOM.
After density modification in shelxe, the contrast of
the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3
for MAD. When I looked at the electron density, the
maps from SAD and MAD are similar, and the solvent
boundary is quite clear. However, the problem is that
the electron density blob passes through the 2-fold
rotation axis, even at 3 rmsd contour level. Also,
the unit cell seems to be too small for the molecule.
I am afraid that the space group assignment is wrong,
but I am a beginner so I nearly have no clue. I did
reprocess the data in P1 and looked at the
self-rotation function with a radius at 200 A. From
the list it seems that there is only one 2-fold
rotation axis. I am quite confused. Could anybody
give me some hint of this problem?
Thanks a lot in advance!
Sincerely,
Chen
--
Nazia Nasir
PhD Scholar
Protein Crystallography Lab
National Institute of Immunology
New Delhi
--
Nazia Nasir
PhD Scholar
Protein Crystallography Lab
National Institute of Immunology
New Delhi
--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582
