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CCP4BB  May 2018

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Subject:

Re: Difficult experimental phasing

From:

"PD Dr. Günter Fritz" <[log in to unmask]>

Reply-To:

PD Dr. Günter Fritz

Date:

Tue, 1 May 2018 14:34:42 +0200

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (36 lines)

Dear Abris,

if you have a partial MolRep solution and  SAD data you might try to 
combine both with phaser.

There is an example script here how to combine the model and SAD data to 
determine and refine the heavy atom substructure:
https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=Phenix&mobileaction=toggle_view_desktop#phenix.phaser_-_SAD_phasing_with_Phaser

You find the same options in the gui and instructions on the official 
PHASER webpage; the script is a bit easier to use if you try different 
things, e.g. combine SAD datasets. This worked well for me with low 
resolution data.

HTH
Guenter
> Dear all,
>
> I’m trying to solve the structure of a putative homodimer, but despite having collected good quality data, it has proven to be rather difficult.
>
> I have collected native and derivative datasets (2.2-4Å, C2 SG, 165/35/115Å//104° unit cell) with acceptable merging statistics. Xtriage, Aimless does not report any apparent twinning, tNCS or significant anisotropy. Based on Matthews coefficient, two monomers should be present in the ASU. Self-rotation function doesn’t give a clear idea on NCS, I see a few weak peaks at Chi=180°. Crystal was checked against contaminant with SDS-PAGE, MS and ContaMiner.
>
> MR approaches so far have failed due to low sequence homology (~20%). I have tried MoRDa, MrBUMP, Balbes or ensembles of different models as homodimer, monomer or divided into smaller domains. I can place the dimer or a monomer with high TFZ score (6-30), but with smaller domains MR usually thrashes. The solution always has a rather high R factor (over 0.5), unclear electron density and does not improve on subsequent intensive rebuilding. Trials in P1 (or in other possible spacegroups) have failed too.
>
> I have recently collected multiple good datasets from a single I3C soaked crystal with measurability extending to 4-7Å on individual datasets or 3-4Å on merged multi-dataset data. No apparent radiation damage or other pathologies present. Moderate isomorphism with native data (<0.4% unit cell difference).
>
> Any attempt to solve the substructure or get interpretable maps has so far failed even with exhaustive SHELX, CRANK2, HySS, Autosol, Phaser or AutoRickshaw runs. MR-SAD or SIRAS approaches have so far failed too. Despite promising HySS solutions (CC>0.35) I can´t see the expected triangle arrangement of the I atoms, can’t discern the hands and it always result in uninterpretable low FOM maps.
>
> I have a few weaker datasets (not as isomorphous though) with different HA soaks (signal up to 7Å), so I could try MIR or multi-crystal averaging, though I have no experience with these approaches.
>
> I can provide more details if needed and I would appreciate any kind of help with my problem.
>
> Thank you very much.
>
> Best regards,
> Abris

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