Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Eleanor Dodson]

This could well be due to radiation damage - S are often affected, also Glu 
and Asp side chains. It is hard to know what to do since the effects are 
time related. If you have high redundancy maybe you could not use he later 
batches? Otherwise maybe just relax the B factor restraints and let them 
show the loss of atoms.. The trouble with that is that you have to relax 
all side-chain B restraints which may not be so appropriate for ILE say...

 Eleanor

On Apr 4 2012, Chris Meier wrote:

MessageDear all, I am refining the X-ray structure of a protein:Data to 
~2A were collected at a latest-generation synchrotron.The 2fo-Fc maps are 
crisp, the model of the protein is complete and I am reasonably happy 
with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I 
am seeing a lot of negative difference density, especially around sulphur 
atoms (negative density around -9 sigma) and oxygen atoms (e.g. 
side-chain oxygens of Glu, Asp, etc. residues with negative density 
around -6 sigma). Has anyone observed this before? I have found CCP4bb 
postings discussing radiation damange of suplphur atoms(e.g. 
http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html 
).Can this also happen with oxygen atoms? What would be an appropriate 
way to deal with this issue during refinement? Suggestions greatly 
appreciated. Thanks,Chris




--
Professor Eleanor Dodson
YSNL, Dept of Chemistry
University of York
Heslington YO10 5YW
tel: 00 44 1904 328259
Fax: 00 44 1904 328266

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Sanishvili, Ruslan]

Hi Chris,
As has been suggested already, and seems quite plausible to me, it sounds like 
tell-tale signs of radiation damage.
To have little more substance behind this suspicion, some more experimental 
details could help:
What was the dose accumulated during data collection?
If the dose cannot be calculated, what was the beam intensity, frame exposure 
time, number of frames, total rotation of the crystal, crystal size, beam size? 
If the beam intensity is not known, the beamline and the attenuation factor 
used might be helpful.

What is the space group and how much data were collected? IF you have data with 
high multiplicity, you may be able to get rid of the latter parts of it 
maintaining completeness. This would reduce the effects of radiation damage if 
you are really dealing with it. Alternatively (but again with highly redundant 
data), you could try "zero dose extrapolation". Look up Kay Diederichs' and 
Dominika Borek's works on this.

Regards,
Nukri

Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris 
Meier
Sent: Wednesday, April 04, 2012 10:16 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] negative difference density around sulphur and oxygen atoms


Dear all,

I am refining the X-ray structure of a protein:
Data to ~2A were collected at a latest-generation synchrotron.
The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).

However, I am seeing a lot of negative difference density,
especially around sulphur atoms (negative density around -9 sigma)
and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
negative density around -6 sigma).

Has anyone observed this before?

I have found CCP4bb postings discussing radiation damange of suplphur atoms
(e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
Can this also happen with oxygen atoms?

What would be an appropriate way to deal with this issue during refinement?

Suggestions greatly appreciated.

Thanks,
Chris

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Katherine Sippel]

On Wed, Apr 4, 2012 at 10:31 AM, Roger Rowlett  wrote:

>  I have also seen a recent paper where radiation damage of a bound protein
> ligand was apparently observed in a synchrotron beam.
>

That was a manuscript were I would have happily given the coordinates and
structure factors to the reviewers with my blessing. Learned a valuable
lesson about adopting orphaned data sets though.

Cheers,
Katherine


> I look forward to hearing from others how best to handle this in
> refinement.
>
> Cheers,
>
> __**_
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>
>
> On 4/4/2012 11:16 AM, Chris Meier wrote:
>
>> Dear all,
>> I am refining the X-ray structure of a protein:
>> Data to ~2A were collected at a latest-generation synchrotron.
>> The 2fo-Fc maps are crisp, the model of the protein is complete and I am
>> reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac
>> 5.5).
>> However, I am seeing a lot of negative difference density,
>> especially around sulphur atoms (negative density around -9 sigma)
>> and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with
>> negative density around -6 sigma).
>> Has anyone observed this before?
>> I have found CCP4bb postings discussing radiation damange of suplphur
>> atoms
>> (e.g. http://www.dl.ac.uk/list-**archive-public/ccp4bb/2004-07/**
>> msg00532.html).
>> Can this also happen with oxygen atoms?
>> What would be an appropriate way to deal with this issue during
>> refinement?
>> Suggestions greatly appreciated.
>> Thanks,
>> Chris
>>
>>
>>

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Yuri Pompeu]

could it be that the scattering table would be slightly different for the 
sulfur atoms at the collected wavelength?
Are they Cys or Met residues? if Cys is there a possibility of oxidation to the 
disulfides?

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Garib N Murshudov]

Dear Chris


Could you please try later version of refmac then if the problem persists 
please let me know. Before making any suggestions it would be good to make sure 
that the problem is not related with particular software version (as Ian 
suggested)


regards
Garib


On 4 Apr 2012, at 16:16, Chris Meier wrote:

> Dear all,
> I am refining the X-ray structure of a protein:
> Data to ~2A were collected at a latest-generation synchrotron.
> The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
> reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
> However, I am seeing a lot of negative difference density, 
> especially around sulphur atoms (negative density around -9 sigma) 
> and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
> negative density around -6 sigma).
> Has anyone observed this before?
> I have found CCP4bb postings discussing radiation damange of suplphur atoms
> (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
> Can this also happen with oxygen atoms?
> What would be an appropriate way to deal with this issue during refinement?
> Suggestions greatly appreciated.
> Thanks,
> Chris
> 

Dr Garib N Murshudov
Group Leader, MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[VAN RAAIJ , MARK JOHAN]

apart from radation damage it could be a combination of: 
- too tight restraints on the B-factors 
- 9 sigma not being that much on a the e/A3 scale, i.e. your difference map is 
very flat (which is good) and the few peaks that remain stand out a lot, even 
if their absolute height is low... 

Quoting Chris Meier:

Message

Dear all,

I am refining the X-ray structure of a protein:
Data to ~2A were collected at a latest-generation synchrotron.
The 2fo-Fc maps are crisp, the model of the protein is complete and I
am reasonably happy with the stats (R below 20%, Rfree below 25% in
Refmac 5.5).

However, I am seeing a lot of negative difference density,
especially around sulphur atoms (negative density around -9 sigma)
and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues
with negative density around -6 sigma).

Has anyone observed this before?

I have found CCP4bb postings discussing radiation damange of suplphur
atoms
(e.g.
http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html
).
Can this also happen with oxygen atoms?

What would be an appropriate way to deal with this issue during
refinement?

Suggestions greatly appreciated.

Thanks,
Chris

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es 

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Ian Tickle]

The PNAS paper you refer to talks about a "loss of definition" of
exposed carboxyl O atoms, i.e. an increase in B factor, but presumably
if this is modelled properly then it shouldn't leave a big hole in the
difference map.  After all, the paper is not claiming that C-O bonds
are broken, only that there is "increased mobility" (or just as
likely, induced static disorder).  I'm wondering if this is related to
too-tight B-factor restraints.  I never use the default settings and
always use more relaxed ones: in particular I set the weights of B
factor restraints across angles to zero, IMO the across-bond
restraints are more than sufficient.  There has been a historical
obsession with getting B factors as low as possible (too-tight
restraints will certainly achieve this if that is your goal!), but
isn't the true goal of refinement to obtain the model which best
explains the data?

Cheers

-- Ian

On 4 April 2012 16:31, Roger Rowlett  wrote:
> PNAS 2000, 97, 623

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Scott Classen]

Hello Chris,

Are you refining individual atomic B factors or grouped? Perhaps the B factors 
of the terminal atoms of the side chain are being restrained to too low of a B 
factor resulting in excessive negative density?

Scott


On Apr 4, 2012, at 8:16 AM, Chris Meier wrote:

> Dear all,
> I am refining the X-ray structure of a protein:
> Data to ~2A were collected at a latest-generation synchrotron.
> The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
> reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
> However, I am seeing a lot of negative difference density, 
> especially around sulphur atoms (negative density around -9 sigma) 
> and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
> negative density around -6 sigma).
> Has anyone observed this before?
> I have found CCP4bb postings discussing radiation damange of suplphur atoms
> (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
> Can this also happen with oxygen atoms?
> What would be an appropriate way to deal with this issue during refinement?
> Suggestions greatly appreciated.
> Thanks,
> Chris
> 

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Jacob Keller]

>
> I look forward to hearing from others how best to handle this in
> refinement.
>
>
Dose-dependent occupancies (tau of an exponential decay function?) refined
against unmerged data

JPK

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Roger Rowlett]

Radiation damage induced loss of definition of disulfide bridges, side 
chain carboxylates, and certain histidine residues has been observed in 
synchrotron-irradiated protein crystals. For example, see Weik et al., 
PNAS 2000, 97, 623. I have also seen a recent paper where radiation 
damage of a bound protein ligand was apparently observed in a 
synchrotron beam.


I look forward to hearing from others how best to handle this in refinement.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 4/4/2012 11:16 AM, Chris Meier wrote:

Dear all,
I am refining the X-ray structure of a protein:
Data to ~2A were collected at a latest-generation synchrotron.
The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
However, I am seeing a lot of negative difference density,
especially around sulphur atoms (negative density around -9 sigma)
and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
negative density around -6 sigma).
Has anyone observed this before?
I have found CCP4bb postings discussing radiation damange of suplphur atoms
(e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
Can this also happen with oxygen atoms?
What would be an appropriate way to deal with this issue during refinement?
Suggestions greatly appreciated.
Thanks,
Chris

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Ian Tickle]

PS you say the model is complete, but just as important how complete
is (are?) the data.

-- Ian

On 4 April 2012 16:16, Chris Meier  wrote:
> Dear all,
> I am refining the X-ray structure of a protein:
> Data to ~2A were collected at a latest-generation synchrotron.
> The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
> reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
> However, I am seeing a lot of negative difference density,
> especially around sulphur atoms (negative density around -9 sigma)
> and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
> negative density around -6 sigma).
> Has anyone observed this before?
> I have found CCP4bb postings discussing radiation damange of suplphur atoms
> (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
> Can this also happen with oxygen atoms?
> What would be an appropriate way to deal with this issue during refinement?
> Suggestions greatly appreciated.
> Thanks,
> Chris
>

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Ian Tickle]

Hi Chris

I would say there's something very wrong if you're seeing -6 sigma
difference peaks at O atoms.  I don't see how this can be explained by
radiation damage.  I for one have never seen that before in a
structure where there weren't other obvious issues (or maybe I just
haven't looked hard enough).

I would try refining it with a different program, e.g. Buster, or even
a different version of Refmac (I use 5.6.x routinely, but I see
there's a 5.7.x now - Garib will no doubt have an opinion on which is
the best one to use).  At least that will eliminate the software as
the origin of the problem: if it doesn't go away then we'll have to
think again.

Cheers

-- Ian

On 4 April 2012 16:16, Chris Meier  wrote:
> Dear all,
> I am refining the X-ray structure of a protein:
> Data to ~2A were collected at a latest-generation synchrotron.
> The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
> reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
> However, I am seeing a lot of negative difference density,
> especially around sulphur atoms (negative density around -9 sigma)
> and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
> negative density around -6 sigma).
> Has anyone observed this before?
> I have found CCP4bb postings discussing radiation damange of suplphur atoms
> (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
> Can this also happen with oxygen atoms?
> What would be an appropriate way to deal with this issue during refinement?
> Suggestions greatly appreciated.
> Thanks,
> Chris
>

[ccp4bb] negative difference density around sulphur and oxygen atoms

[Chris Meier]

Dear all,
I am refining the X-ray structure of a protein:
Data to ~2A were collected at a latest-generation synchrotron.
The 2fo-Fc maps are crisp, the model of the protein is complete and I am 
reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5).
However, I am seeing a lot of negative difference density, 
especially around sulphur atoms (negative density around -9 sigma) 
and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with 
negative density around -6 sigma).
Has anyone observed this before?
I have found CCP4bb postings discussing radiation damange of suplphur atoms
(e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).
Can this also happen with oxygen atoms?
What would be an appropriate way to deal with this issue during refinement?
Suggestions greatly appreciated.
Thanks,
Chris