Sorry, I'm coming a bit late to this thread, and most things I would have suggested have already been covered by Tom and Paul.  Certainly there are examples in the literature of people using MR to place a heavy atom substructure against the anomalous differences, but as Tom pointed out there's the complication of making sure that you get the Se sites from one molecule and not from a combination of symmetry-related molecules, which probably won't have the same relative arrangement in the other crystal form.

The one remaining thing I'd like to explore is whether you're sure that you've carried out the molecular replacement with density properly.  This has become easier (with Tom's phenix.cut_out_density program), but it's still relatively complicated and error-prone.  First, you have to be sure that you've cut out the density essentially for one molecule, and not a mixture of symmetry-related molecules.  If the location and rough shape of the molecule isn't clear in your map, you may have to try things like cutting out spheres of density from different places in the unit cell and seeing which gives the clearest MR solution.  Second, you have to make sure that you place the density in a reasonably large unit cell, 2.5-4 times the extent of the density that you've cut out, before using it as an MR model.  Third, you have to choose an appropriate value for the effective RMS error of the density model.  If your phases go to 4A, then a number like 1.5A to 2A might be appropriate.

In our tests, the map can be pretty noisy but you can still get a very clear signal from the MR search.  Of course, if the phase information is very weak, it still may not work even with an optimal envelope.

Best wishes,

Randy Read

On 23 Jun 2013, at 15:56, "Terwilliger, Thomas C" <[email protected]> wrote:

You could try both the isomorphous and the anomalous differences.  Each one has some useful information. You can even combine them but I'm guessing it won't help too much. 

Paul Adams' suggestion to check out related space groups is a very good one. Phenix.autosol only tries the opposite hand, so you have to try all the different screw axis possibilities that are not related by inversion.

Another thing you could try is to repeat your MR with density from your map (against the 2.6 A native) and get a long list of solutions. For each solution take the phases after MR and use them to calculate an anomalous difference Fourier for your semet P2221 data.  Then notice if there are peaks in this map, and whether these peaks have the same relationship to the MR-placed density as you saw in your original map.  If yes, you may have a solution.

All the best,
Tom T

From: [email protected] [[email protected]] on behalf of zhaoy [[email protected]]
Sent: Sunday, June 23, 2013 3:32 PM
To: PHENIX user mailing list
Subject: Re: [phenixbb] what about heavy atom as model for MR

Thank you very much.  I will try as you said.

 

The P2221 dataset's qulity are not very well. The points from diffraction image are not sharp. Besides, native dataset is 2.6A, setmet anomalous datasets is only 4A. So I have not obtained the solution with p2221datasets.

There is only one molecular per AUS at p4322. So, from the initial map, I can confirm the heavy atom sites from the same molecular.

Maybe I have a general idea about the method you said. But I still puzzled which F difference should I use, isomorphous or anomalous differences?

 

Thanks!

--

Yan Zhao, M.Phil.
National Laboratory of Protein Sciences
Institute of Biophysics
Chinese Academy of Sciences
15 Datun Rd.
Beijing, 100101
China
 
Terwilliger, Thomas C 写:
I'm not sure if there is a tool that will create the file you want for this.  You could however just write a little script that reads in a .sca file and calculates F from I, subtract F+ - F- to get Dano, square that, write out Dano**2 and sigma(Dano**2) as another .sca file.  Then you could use this as input to molecular replacement with your sites from the P4322 dataset. 

There is an important caveat to this approach however:  your sites from the P4322 have to be all part of the same molecule, and this might or might not happen automatically.  If some sites are from one molecule and others from another molecule, then in the other crystal form their relationship won't be correct.  If there is only one molecule in the au then this might or might not be a big problem. If there are more than one it is very likely to be a big problem.   This would apply to the density search that you carried out as well.  If your density is good enough to see where the molecule is, you might be able to figure out which of the symmetry-related sites to include in your MR search.

Nevertheless as this is easy it seems like a fine thing to do.  However I am wondering if it will work because your 2.6 A semet anomalous dataset did not yield a solution by itself, which is a little surprising...  Also of course if your P4322 solution is not actually right or even just not close enough it would not work.

-Tom T

From: [email protected] [[email protected]] on behalf of 赵岩 [[email protected]]
Sent: Sunday, June 23, 2013 7:52 AM
To: [email protected]
Subject: [phenixbb] what about heavy atom as model for MR

Hi everyone,

 

The heavy atoms(Se-Met) were located at P4322 space group(4A). But the initial density map is so bad that I can not build the initial model.

 

Meanwhile, I have another two datasets, native(2.6A) and  se-met anomalous dataset with space group P2221. But these two datasets have no solution. I have tried the initial map from sp P4322 as a model to do molecular  replacement. But no MR solution. 

So, I want to know how can I use the heavy atom as model to do MR with isomorphous or anomalous differences?

 

Thanks!

--

Yan Zhao, M.Phil.
National Laboratory of Protein Sciences
Institute of Biophysics
Chinese Academy of Sciences
15 Datun Rd.
Beijing, 100101
China
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Randy J. Read
Department of Haematology, University of Cambridge
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