[phenixbb] DTT mediated disulfide crosslink across a crystallographic two-fold! (Pavel Afonine)
Joseph Noel
noel at salk.edu
Wed Aug 4 10:43:27 PDT 2010
Thanks Pavel! I will wait and see what Ralf says regarding defining the symmetry operator. If I am using the GUI for phenix.refine should I create a new file with the bond definition you mention and include it as one of the starting files or should I edit the Refine_*.eff file that gets created and insert it somewhere there?
___________________________________________________________
Joseph P. Noel, Ph.D.
Investigator, Howard Hughes Medical Institute
Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics
The Salk Institute for Biological Studies
10010 North Torrey Pines Road
La Jolla, CA 92037 USA
Phone: (858) 453-4100 extension 1442
Cell: (858) 349-4700
Fax: (858) 597-0855
E-mail: noel at salk.edu
Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37
Web Site (HHMI): http://hhmi.org/research/investigators/noel.html
___________________________________________________________
On Aug 4, 2010, at 10:22 AM, phenixbb-request at phenix-online.org wrote:
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> Today's Topics:
>
> 1. Re: DTT mediated disulfide crosslink across a
> crystallographic two-fold! (Joseph Noel)
> 2. Re: high average b-factor vs. Wilson B (Gino Cingolani)
> 3. Re: high average b-factor vs. Wilson B (Frank von Delft)
> 4. Re: DTT mediated disulfide crosslink across a
> crystallographic two-fold! (Pavel Afonine)
> 5. Re: high average b-factor vs. Wilson B (Pavel Afonine)
> 6. r-work and r-free for specific resolution range(s)
> (Kay Diederichs)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 4 Aug 2010 04:53:25 -0700
> From: Joseph Noel <noel at salk.edu>
> To: phenixbb at phenix-online.org
> Subject: Re: [phenixbb] DTT mediated disulfide crosslink across a
> crystallographic two-fold!
> Message-ID: <91F9FC5E-00E4-454C-AB99-2DD353BDC4A5 at salk.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi Ralf and Pavel,
>
> Unfortunately, Phenix didn't recognize a disulfide bond should exist between the sulfur atoms of DTT and the side chain sulfur atom of the cysteine residue in question. I position everything well in the Fo-Fc and 2Fo-Fc maps and ensured the two sulfurs were approximately 2.1 A apart. However, after refinement the DTT molecule moved so the two sulfurs in question where nearly 3 A away. Maybe this is because it is a mixed disulfide between a DTT molecule and a side-chain Cys rather then what most programs expect which is a disulfide between two Cys residues. Is it possible I need to define the complete chemical entity as a hetatm (the disulfide linked Cys residue? Is there a way to specify that a covalent bond should exist between the SG atom of the Cys in question and the S atoms of DTT with DTT lying across the crystallographic two-fold given its internal 2-fold symmetry?
>
> Joe
> ___________________________________________________________
> Joseph P. Noel, Ph.D.
> Investigator, Howard Hughes Medical Institute
> Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics
> The Salk Institute for Biological Studies
> 10010 North Torrey Pines Road
> La Jolla, CA 92037 USA
>
> Phone: (858) 453-4100 extension 1442
> Cell: (858) 349-4700
> Fax: (858) 597-0855
> E-mail: noel at salk.edu
>
> Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37
> Web Site (HHMI): http://hhmi.org/research/investigators/noel.html
> ___________________________________________________________
>
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> ------------------------------
>
> Message: 2
> Date: Wed, 4 Aug 2010 08:36:46 -0400 (EDT)
> From: Gino Cingolani <Gino.Cingolani at jefferson.edu>
> To: "PHENIX user mailing list" <phenixbb at phenix-online.org>
> Subject: Re: [phenixbb] high average b-factor vs. Wilson B
> Message-ID: <20100804083646.AMK95477 at logan.jefferson.edu>
> Content-Type: text/plain; charset=us-ascii
>
> Dear Geoffrey,
>
> I had similar issues on a lower resolution structure, which Phenix kept refining to a B-factor >120-150A2, despite my average Wilson B-factor was (only) 85A2.
>
> I am aware of all approximation made in defining the Wilson B-factor and how one should not take the Wilson B-factor too literally (etc.. etc...). But, as a matter of fact, Phenix kept over-refining my B-factor to crazy values. And I have seen in
> two cases, both with VERY large structures with >15,000 residues/asym unit and ncs ranging between 4- to 24-fold.
> So I admit these structures may not be your everyday refinement benchmark. At any rate, after several months of troubleshooting here what I can suggest:
>
> FIRST
>
> 1. Was your diffraction anisotropic? If so, try the
> Diffraction Anisotropy Server. The server applies a negative isotropic B-factor correction to your data. In my case, corrected structure factor amplitudes resulted in a 2.5% drop in the Rfree and the average B-factor of my structure (which phenix routinely refined to >120-150A2 after only 3 macrocycles) went down to ~90A2, quite close to my Wilson B-factor.
> More importantly, when I use 'corrected' data, I can refine
> even with 10 macrocycles and my structure B-factor stays constant.
>
> SECOND
> How many macrocycles of refinement are you using?
> In my case Phenix kept bumping up the average B-factor of my structure by ~10A2 macrocycle. A 10 macrocycles refinement would bring the averaged B-factor to ~200A2. So don't over-refine.
> Stick to 2-3 cycles (provided your geometry is good, of course).
>
> THIRD
> Do you have NCS and are you using TLS?
> If so, try to first use NCS to refine atom positions,
> then do TLS refinement without imposing NCS.
> If you think about, it doesn't make too much sense to use ncs-restraints when splitting a structure in TLS groups.
>
> FOURTH
> Are you playing with weights (say ... wxc_scale=0.5 and wxu_scale=1) and do you have NCS? If so, tightening the geometry too much in the presence of NCS can bump-up our B-factor.
> Inspect your Phenix output file to look at the rms deviations among your ncs-copies in the asymmetric unit.
>
> Good luck!
>
> Gino
>
> ******************************************************************************
> Gino Cingolani, Ph.D.
> Associate Professor
> Thomas Jefferson University
> Dept. of Biochemistry & Molecular Biology
> 233 South 10th Street - Room 826
> Philadelphia PA 19107
> Office (215) 503 4573
> Lab (215) 503 4595
> Fax (215) 923 2117
> E-mail: gino.cingolani at jefferson.edu
> ******************************************************************************
> "Nati non foste per viver come bruti, ma per seguir virtute e canoscenza"
> ("You were not born to live like brutes, but to follow virtue and knowledge")
> Dante, The Divine Comedy (Inferno, XXVI, vv. 119-120)
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 04 Aug 2010 14:00:05 +0100
> From: Frank von Delft <frank.vondelft at sgc.ox.ac.uk>
> To: Gino.Cingolani at jefferson.edu, PHENIX user mailing list
> <phenixbb at phenix-online.org>
> Subject: Re: [phenixbb] high average b-factor vs. Wilson B
> Message-ID: <4C596455.3040002 at sgc.ox.ac.uk>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
>
>
> On 04/08/2010 13:36, Gino Cingolani wrote:
>> THIRD
>> Do you have NCS and are you using TLS?
>> If so, try to first use NCS to refine atom positions,
>> then do TLS refinement without imposing NCS.
>> If you think about, it doesn't make too much sense to use ncs-restraints when splitting a structure in TLS groups.
>>
> I assume you're talking specifically about NCS B-factor restraints?
> That I'd agree with -- indeed, I routinely loosen NCS B-factor
> restraints even without TLS>
>
> However if what you're recommending is to remove NCS xyz-restraints as
> well, only because you're including TLS groups refined, I'm afraid that
> does not make sense: TLS is part of the uncertainty parametrization,
> not of the positional parameters.
> phx.
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 04 Aug 2010 08:07:53 -0700
> From: Pavel Afonine <pafonine at lbl.gov>
> To: phenixbb at phenix-online.org
> Subject: Re: [phenixbb] DTT mediated disulfide crosslink across a
> crystallographic two-fold!
> Message-ID: <4C598249.2070802 at lbl.gov>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi Joe,
>
> Ralf correct me if there is a better way of doing this...
> You can define a bond between any selected atoms:
>
> http://www.phenix-online.org/documentation/refinement.htm#anch84
>
> For example:
>
> refinement.geometry_restraints.edits {
> excessive_bond_distance_limit = 10
> bond {
> action = *add delete change
> atom_selection_1 = chain A and resseq 123 and name SG
> atom_selection_2 = chain X and resseq 345 and name S1
> symmetry_operation = None
> distance_ideal = 2.1
> sigma = 0.02
> slack = None
> }
> }
>
> It is important in your case that you need to define a symmetry
> operation that relates both atoms in question. There is a trick to get
> it in Coot but I forgot (Ralf knows - he showed me at some point) - I'm
> sure it was on phenixbb a few times in the past.
>
> After this everything should work.
>
> Pavel.
>
>
>
> On 8/4/10 4:53 AM, Joseph Noel wrote:
>> Hi Ralf and Pavel,
>>
>> Unfortunately, Phenix didn't recognize a disulfide bond should exist
>> between the sulfur atoms of DTT and the side chain sulfur atom of the
>> cysteine residue in question. I position everything well in the Fo-Fc
>> and 2Fo-Fc maps and ensured the two sulfurs were approximately 2.1 A
>> apart. However, after refinement the DTT molecule moved so the two
>> sulfurs in question where nearly 3 A away. Maybe this is because it is
>> a mixed disulfide between a DTT molecule and a side-chain Cys rather
>> then what most programs expect which is a disulfide between two Cys
>> residues. Is it possible I need to define the complete chemical entity
>> as a hetatm (the disulfide linked Cys residue? Is there a way to
>> specify that a covalent bond should exist between the SG atom of the
>> Cys in question and the S atoms of DTT with DTT lying across the
>> crystallographic two-fold given its internal 2-fold symmetry?
>>
>> Joe
>
>
>
> ------------------------------
>
> Message: 5
> Date: Wed, 04 Aug 2010 08:28:31 -0700
> From: Pavel Afonine <pafonine at lbl.gov>
> To: Gino.Cingolani at jefferson.edu, PHENIX user mailing list
> <phenixbb at phenix-online.org>
> Subject: Re: [phenixbb] high average b-factor vs. Wilson B
> Message-ID: <4C59871F.8050503 at lbl.gov>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi Gino,
>
> just a few comments:
>
>> I am aware of all approximation made in defining the Wilson B-factor and how one should not take the Wilson B-factor too literally (etc.. etc...). But, as a matter of fact, Phenix kept over-refining my B-factor to crazy values.
>
> I would say refining and not over-refining, because nobody knows where
> "over" starts.
>
>> 1. Was your diffraction anisotropic? If so, try the
>> Diffraction Anisotropy Server. The server applies a negative isotropic B-factor correction to your data. In my case, corrected structure factor amplitudes resulted in a 2.5% drop in the Rfree
>
> That server removes reflections by sigma value and in selected
> directions. In some cases it can easily be a few thousands of Fobs. So,
> it it not surprising that the R-factors dropped. You can't compare the
> R-factors for the datasets containing different amount of reflections.
>
>> and the average B-factor of my structure
>
> and it is not surprising that the B-factor dropped: instead of keeping
> it in ATOM records you gave it away to uncle Sam, by applying negative-B
> correction. Just a trade, matter of where you keep these extra B-factor
> value.
>
>> SECOND
>> How many macrocycles of refinement are you using?
>> In my case Phenix kept bumping up the average B-factor of my structure by ~10A2 macrocycle. A 10 macrocycles refinement would bring the averaged B-factor to ~200A2. So don't over-refine.
>> Stick to 2-3 cycles (provided your geometry is good, of course).
>
> Interesting. I've never seen it before. Did you try to change the target
> weights for ADP refinement: manually or automatically? If you did and
> none helped (although I'm still not sure if help is needed) I would be
> interested to see the data and model.
>
>> THIRD
>> Do you have NCS and are you using TLS?
>> If so, try to first use NCS to refine atom positions,
>> then do TLS refinement without imposing NCS.
>> If you think about, it doesn't make too much sense to use ncs-restraints when splitting a structure in TLS groups.
>
> Regarding NCS: in ADP refinement it is applied only to local ADPs, and
> not total ones. See recent publication regarding this in PHENIX newsletter:
> http://www.phenix-online.org/newsletter/CCN_2010_07.pdf
> see page 28.
>
> So you can safely refine everything in one go (I guess).
>
> All the best!
> Pavel.
>
>
>
> ------------------------------
>
> Message: 6
> Date: Wed, 04 Aug 2010 19:22:08 +0200
> From: Kay Diederichs <kay.diederichs at uni-konstanz.de>
> To: phenixbb at phenix-online.org
> Subject: [phenixbb] r-work and r-free for specific resolution range(s)
> Message-ID: <4C59A1C0.9050505 at uni-konstanz.de>
> Content-Type: text/plain; charset="iso-8859-15"; Format="flowed"
>
> Hi,
>
> is there a way to get the R-factor statistics from phenix.refine, for a
> specific resolution range, e.g. 2.5-2.6A ? The reason I ask is that I
> would like to specify the same resolution range as the data reduction
> program does.
>
> I tried with
> phenix.reflection_statistics x_map_coeffs.mtz x_data.mtz
> --low-resolution=2.6 --resolution=2.5
>
> but with the default settings, phenix.refine writes 2FOFCWT and FOFCWT,
> but not FC . So it looks like I have to re-run phenix.refine and make it
> write out FC? Or is it easier to run phenix.xmanip for that purpose?
>
> thanks,
>
> Kay
>
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