Re: [phenixbb] ENSEmble parameters when the model is an EM map

Dear Peter, On the first point, it shouldn't be necessary to worry about the overall scale and temperature factor, because Phaser works with normalized data (from which the overall scale and B have been removed). This effectively makes the average intensity as a function of resolution the same for the EM map and the X-ray data. On the other hand, you have to give Phaser an effective RMS error (i.e. the RMS error that would give the same structure factor error for coordinates as you expect for the EM image) -- I think a value of 1/3 to 1/2 of the resolution of the EM image would be a good enough guess. This is needed to calibrate the likelihood targets, so that it is known how well the observed and calculated structure factors should agree for the correct solution. I didn't realize that the EMDB doesn't check whether the magnification is given in the deposition. That's an issue that should be addressed. If you're talking about cryo-EM data, where you get a 3D density distribution, then I don't think that cut-off levels should be an issue. If it's negative stain data, then that would be a different story. However, I don't have a good feel for how well negative stain images can be used for MR. If anyone has any experience, it would be good to hear about it! As for the origin and the extent, these don't have to be particularly precise, so you could just approximate them by visual inspection of the EM map. The extent is used to compute how large the rotation step can be (i.e. how much you can rotate before the edge of the object rotates a large amount relative to the resolution of the data), and that rotation is carried out around the centre that you specify. Also, the quality of the interpolation depends on getting these approximately right. Roughly, the cell into which you put the object has to be large relative to the furthest distance of a point in the object from the specified centre, so if you specify the centre badly, you would need a larger cell to get accurate interpolation. Best wishes, Randy Read On 23 Jul 2009, at 19:08, Peter Grey wrote:

Dear Prof. Read,

Thank you for the swift and kind reply. I would like to mention than there are several difficult issues in using EM maps. For example it would be wise to use a temperature factor to sharpen/blur the map so that the histogram of amplitudes vs. resolution would look similar to that of the measured structure factors (better for MR). In addition one needs to determine visually the cut-off level for the mask. Visual inspection is also required to determine the magnification because many maps are deposited in the EMDB with absolutely arbitrary magnification. EMDB is not run like PDB and standartization is not really practiced (sadly) - All these parameters should have been given by the microscopist.

I am familiar with the Phaser example you mentioned but there the problem of finding the origin and extent is easy because you start from a pdb file (to obtain the mask). The approach I suggested is very primitive. Is there a program that can determine more precisely the "box" that contains the particle and its centre of mass ? It should be easy becasue at this stage you already have the mask (whose cut-off was determined by visual inspection) that dictates what is "particle" and what is not in the map.

Peter.

On Thu, Jul 23, 2009 at 5:51 PM, Randy Read wrote: Dear Peter,

There's a new Phenix GUI for Phaser that's under development, and when that 's finished you'll be able to run all possible Phaser jobs from the GUI. In the meantime, it's probably easiest to use command scripts.

A number of the practical issues of using EM maps are the same as using density cut out from an X-ray electron density map, e.g. making sure the cell is big enough (so that the interpolation of the transform of the density works) and knowing where the centre of the object is. These are discussed on our web page, at http://www-structmed.cimr.cam.ac.uk/phaser/density_as_model.html .

Assuming that your large P1 cell is at least 2.5 times as big in all directions as the small cell, then your approach sounds reasonable.

For EM maps, there's an extra issue. It seems that there is generally an uncertainty of several percent in the magnification factor, so you will want to adjust that up and down -- steps of 0.5% would probably be fine enough. The easiest way to do this is to scale the cell dimensions in the MTZ file containing the structure factors derived from the EM map. You could do that easily in sftools, and then run searches with all the scaled MTZ files.

Good luck! I'd be interested in hearing how you get on, and don't hesitate to ask any further questions.

Best wishes,

Randy Read

On 23 Jul 2009, at 16:08, Peter Grey wrote:

...

Dear benevolent experts,

I am trying to use Phaser for molecular replacement with EM-map as a model. The EM map, originally in a small P1 cell, was put in large P1 using CCP4's MAPROT and a mask that was defined by a certain cut- off level. Could you please advise me how to derive the EXTEnt and CENTre parameters needed for ENSEmble building ? Does the simple way of giving the size of the original P1 as the EXTEnt and its centre as CENTre sound reasonable ?

I would be grateful for any advice and comments,

Peter.

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------ Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www- structmed.cimr.cam.ac.uk

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------ Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www- structmed.cimr.cam.ac.uk