Re: [phenixbb] anomalous difference map calculation

Hi Alexandra, When you’re completing a substructure, then we find that it is best to do a different search for each element type, as Tom suggests. However, we found that when you have a basically complete model and you’re trying to identify both the anomaous scatterers and what kind of atom they are, things are a bit different, because there’s already a model for the real part of the scattering of all the atoms. In the ion identification algorithm (a special case of this problem), which we and others helped Nat Echols to develop (http://journals.iucr.org/d/issues/2014/04/00/lv5059/lv5059.pdf), log-likelihood-gradient maps are computed with Phaser by looking for purely imaginary scatterers (special atom type AX, with f+f’=0, f”=1). If you get a significant peak, it almost certainly identifies an anomalous scatterer, and when the occupancy of the purely imaginary scatterer is refined, what that represents is the product of the occupancy for the actual atom and the f” for that atom. Depending on how good the anomalous signal is (which will determine how accurate that refined occupancy is), that can really help to narrow down the identity of an atom. Best wishes, Randy Read ----- 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

On 17 Jun 2015, at 18:32, Terwilliger, Thomas Charles wrote:

I forgot to mention if you use the LLG maps in Phaser, you select the elements that you are looking for and the wavelength of data collection. This is a really powerful feature because it allows Phaser to specifically look for each of the elements you are interested in (and the answers will be different for each because the f' and f" will be different for the different elements).

All the best, Tom T

From: Terwilliger, Thomas Charles Sent: Wednesday, June 17, 2015 11:21 AM To: Alexandra Marques Cc: [email protected]; Terwilliger, Thomas Charles Subject: RE: [phenixbb] anomalous difference map calculation

Hi Alexandra,

I think your refinements without the anomalous differences are going to be very similar to those with the anomalous differences, so probably no it is not worth it to go back. However it is worth making sure that you are using the same test set (R_free_flags) throughout.

If you want to get the very best anomalous map possible, then you could use instead Phaser to calculate an LLG map for the anomalous differences. You provide your refined model (without the anomalously-scattering atoms) and your anomalous data and it can calculate this map for you.

To get the LLG map, in the Phenix GUI go to Experimental Phasing and select Phaser-EP. Then select the Phaser mode "MR-SAD phasing". Then put in your model and anomalous data. Then under "Other Settings" scroll down and check "Output LLG maps". Then run and that should do it! Airlie or Randy might have further comments on this!

All the best, Tom T

From: [email protected] [[email protected]] on behalf of Alexandra Marques [[email protected]] Sent: Wednesday, June 17, 2015 11:07 AM Cc: [email protected] Subject: Re: [phenixbb] anomalous difference map calculation

Thank you very much for your replies.

Here is what I did:

1)I rerun xdsconv again with Friedls law=false in order to get the mtz file with the F(+) and F(-) columns (new.mtz).

2) I used my pdb file from the last refinement together with the new.mtz file as input for “Calculate maps” in Phenix and selected the option for anomalous map.

Now I obtained the ANOM PHANOM map. I hope this is correct…

In the anomalous map the largest density is in the Mo and Fe atoms. There is no density in the S of SO3 or SO4 groups, so, this will not help me to decide whether there are SO3 groups in the structure (because for the SO4 I have no doubt).

However, while searching the mailling list I foud this post http://www.phenix-online.org/pipermail/phenixbb/2009-September/013799.html which worried me. Should I have used from the beginning this “new” mtz file with F+/F- in order to obtain anomalous differences map at each refinement step? Do you think that I should go back, transfer the R-free flags to my new mtz file and repeat all the refinements?

Best regards, Alexandra

2015-06-17 16:59 GMT+01:00 Pavel Afonine : Hi Alexandra,

to get anomalous difference map you do not need to do anything special: phenix.refine calculates this map by default as long as your input data are Fobs(+) and Fobs(-) or Iobs(+) and Iobs(-).

Refining occupancy of Mo may not be a bad idea given it is likely to be partially occupied.

Pavel

On 6/17/15 06:04, Alexandra Marques wrote:

...

Hi,

I am in the last refinement steps of a MR model and I want to calculate an anomalous difference map essentially to confirm the presence of a sulfite molecule and to locate vanadium (present in soaking solution). I read that it is necessary to have a mtz file with anomalous data (i.e. F+,F- or I+,I-). However, my data was collected at “normal” wavelenght (0.97) and it was processed with XDS considering Friedls law= true and my mtz file contain the following columns: H K L FP SIGFP. So, can I still calculate a anomalous difference map based on my data?

Since I also have a Mo atom in the active site can I try to refine its occupancy by using the option “anomalous groups” in the refinement strategy?

Thank you very much,

Alexandra

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