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CCP4BB  February 2012

CCP4BB February 2012

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Subject:

Re: Extra positive density seen after TLS refinement?

From:

Horacio Botti <[log in to unmask]>

Reply-To:

[log in to unmask][log in to unmask]

Date:

Fri, 24 Feb 2012 10:56:47 -0300

Content-Type:

text/plain

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Dear Naveed A Nadvi

I think that your results highlight the fact that modelling the  
disorder/complex ordering of your crystal is relevant and in general,  
that we should take care in optimizing B factor refinement as a strong  
factor for model improvement.

In this sense I would not relay in one TLS group definition, even  
though you have obtain better results comparing no TLS vs TLS. Try  
common sense definitions also: does the protein have a hinge between  
two domains, use the domains as TLS groups, etc. Then, look for  
optimal NCS between TLS groups also.

Best wishes

Horacio





Quoting Naveed Ahmed Nadvi <[log in to unmask]>:

> Dear Crystallographers,
>
> Thank you for your responses. The density map levels were 0.11e/A^3   
> (3.00 A) for both images with and without TLS refinement. When I   
> superposed the deposited structure I could see the extra density was  
>  due to water moleucles that were seen in the higher resolution   
> deposited structure. It is so interesting how I could not see them   
> in my data without doing TLS.
>
> Performing the TLS refinement improved overall parameters:
>
>                                         no TLS/TLS
> R                                    0.2425/0.2334
> R-free                             0.2809/0.2748
> RMS BondLen               0.0092/0.0074
> RMS BondAngle            1.1812/1.1407
> ChirVol                          0.0806/0.0779
>
> My question is, do the positive density seen after TLS refinement   
> justify adding these solvent molecules especially when they were not  
>  observed without TLS refinement?
>
> Thank you once again for your insights!
>
> Naveed
>
> ________________________________________
> From: Ethan Merritt [[log in to unmask]]
> Sent: Sunday, 19 February 2012 3:10 PM
> To: Naveed Ahmed Nadvi
> Cc: [log in to unmask]
> Subject: Re: [ccp4bb] Extra positive density seen after TLS refinement?
>
> On Saturday, 18 February 2012, Naveed A Nadvi wrote:
>> Dear crystallographers,
>>
>>
>>
>> I am fairly new in crystallographic work and structure   
>> determination, but I thought this would be the best place to post   
>> my questions. We had collected structural data for a protein that   
>> diffracted to 3 A. We had used a previously deposited structure   
>> (1.5 A) for molecular replacement. Our final structure used NCS   
>> restraints refinement over 4 chains within the assymetric unit. We   
>> were able to assign some water moleules using COOT and subsequently  
>>  removed 'bad waters' manually. We used automated settings when   
>> dealing with these water molecules. In all cases these water   
>> molecules were found in the same positions as the initial structure  
>>  (1.5 A) that we had used as a search model. This gave us  
>> confidence  in the placement of our water molecules. Finally we had  
>> run  validation tools (MolProbity) and our structure was found to  
>> be  with Molprobity score within the 100th percentile.
>>
>>
>>
>> We then performed a TLS refinement (from TLSMD) to further improve   
>> R values. We used the final MolProbity-validated structure using 8   
>> TLS groups and using PureTLS with constant B factor (50). We are   
>> observing large positive densities from the subsequent REFMAC5   
>> refinement that are otherwise not observed in the absence of TLS   
>> refinement.
>
> Is it possible that the peaks are not higher in terms of absolute   
> electron level,
> but only in terms of RMSD?   That is, if the TLS treatment cleans up the map
> everywhere, then whatever peaks are left will deviate more from the   
> (now lower)
> mean value even though their absolute size is the same.
> In other words, the "3 sigma" contours in your first map may be more like
> 6 sigma contours in your second (cleaner) map.
>
>> My questions are:
>>
>> 1) Is TLS suitable for our dataset (3 A)?
>
> There is no universal answer to that.  You just have to test for   
> yourself each time.
> Certainly TLS can help a lot at 3A for some structures.  In general the more
> anisotropy is present, the more it helps to include it in your model  
>  somehow -
> and TLS is a "cheap" way to include it in your model.  But if your   
> structure does
> not have much anisotropy, then adding TLS to describe it won't have   
> much effect.
>
>> 2) Is TLS refinement independent of NCS refinement or should I   
>> define my NCS based on the 8 TLS groups?
>
> They are not the same thing at all.
>
>> 3) Is it normal to see extra positive density after TLS refinement   
>> and what does it mean?
>
> See possible explanation above.
>
>         Ethan
>
>
>> 4) We had PEG4000 and Tris in our crystallization buffer. Could   
>> these 'blobs' represent these molecules or short water chains? I   
>> have attached images of the largest blob.
>>
>>
>>
>> Any comments and suggestions would be highly appreciated.
>>
>>
>>
>> Kind regards,
>>
>>
>>
>> Naveed A Nadvi
>>
>>
>>
>> Faculty of Pharmacy,
>>
>> University of Sydney, Australia
>>
>>

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