Re: [ccp4bb] off-topic: structural motif / domain comparison

[Chris Fage]

Hi Sam,

Perhaps the Modeller service is what you’re looking for?

https://salilab.org/modeller/

Best wishes,
Chris


On Fri, 6 Aug 2021 at 07:42 Sam Tang  wrote:

> Dear all
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any search program which allows one to 'blast' a protein domain just like
> we 'blast' a protein sequence? For example I have an epitope in hand and
> would like to find out whether this also exists in other proteins. Most
> programs I accessed are based on sequence similarity but is there any
> program which searches a structure against a database of structures?
>
> BRs
>
> Sam
>
> --
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Re: [ccp4bb] Covalent Cysteine Aduct

[Chris Fage]

Hi Richard,

I recently observed the sulfenic acid derivative of a cysteine residue
(S-hydroxy-Cys, ligand ID CSO) in one of my high-resolution maps. Could it
be possibly be this, H-bonded to a nearby water molecule?

Best wishes,
Chris


On Fri, Mar 27, 2020 at 21:33 Paul Emsley  wrote:

>
> On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:
>
>
>
>   although [BME] seems unlikely, since the crystallized protein is a Fab.
>
>
> I don't follow.
>
>
>
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Re: [ccp4bb] Unusual monomer-monomer interface in crystal

[Chris Fage]

Many thanks for all of the suggestions. Ni2+ or Zn2+ seem to be the most
likely culprits, but I plan to carry out a fluorescence scan at my next
beam session.

While it’s interesting that others have observed crystallization-driven
formation of disulfides, the binding of a metal ion at the given position
looks much more probable.

Best wishes,
Chris


On Wed, Jan 22, 2020 at 17:56 Sarah Bowman  wrote:

> This conversation brings up the question of whether it would be good
> practice to always do a fluorescence scan of a crystal sample, whether it
> is known to be a metalloprotein or not...
>
>
>
> Energy dispersive X-ray fluorescence spectra (accessible at most
> synchrotron sources at this point) can often tell you whether there is
> metal in your sample and, because the transitions are element specific,
> what the metal identity is (although it can't place the metal - you need
> the anomalous difference for that).
>
>
>
> Note also there is a recent paper out in JACS using PIXE to investigate
> metalloproteins in the PDB which resolved a number of misidentified or
> missing metal atoms in the structures: High-Throughput PIXE as an Essential
> Quantitative Assay for Accurate Metalloprotein Structural Analysis:
> Development and Application J. Am. Chem. Soc. 2020, 142, 1, 185-197
> https://doi.org/10.1021/jacs.9b09186
>
>
>
> Metals are everywhere!!
>
>
>
> Cheers,
>
> Sarah
>
>
>
> *Sarah EJ Bowman, PhD*
>
>
>
> Associate Research Scientist, Hauptman-Woodward Medical Research Institute
>
> Director, High-Throughput Crystallization Screening Center
>
> Research Associate Professor, Department of Biochemistry, University at
> Buffalo
>
>
>
> Research Webpage <https://hwi.buffalo.edu/scientist-directory/sbowman/>
>
> www.getacrystal.org
>
>
>
> sbow...@hwi.buffalo.edu
> 716-898-8623
>
>
>
>
>
> *From: *CCP4 bulletin board  on behalf of
> Christian Roth 
>
>
> *Reply-To: *Christian Roth 
> *Date: *Wednesday, January 22, 2020 at 12:17 PM
> *To: *"CCP4BB@JISCMAIL.AC.UK" 
> *Subject: *Re: Unusual monomer-monomer interface in crystal
>
>
>
> Hi Chris,
>
>
>
> it could be all Ni besides Zn. I've seen Ni carried over from the initial
> metal affinity chromatography I presume.
>
>
>
> Cheers Christian
>
>
>
> Chris Fage  schrieb am Di., 21. Jan. 2020, 21:23:
>
> Thanks to Guenter and Eleanor for their replies. I mentioned that there is
> not adequate space for a metal ion at the described interfaces.
> Nevertheless, placement of a metal ion, followed by refinement in Phenix,
> repositions the side chains significantly so as to make room for the ion
> without distorting geometry. There is also a very strong difference signal
> centered between the four side chains. This agrees with the MS data, which
> indicate a monomeric state without a disulfide linkage. Now, I just need to
> identify the metal. A Zn2+ seems to fit well based on coordination number
> and interatomic distances, as Guenter exemplified.
>
>
>
> Best wishes,
>
> Chris
>
>
>
> On Tue, Jan 21, 2020 at 6:33 PM Eleanor Dodson <
> 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> Easy to check whether it is a metal by looking at an anomalous difference
> map..
>
> But there are examples of di-sulphides formed between symmetry related
> molecules..
>
> Query the wwwpdb -
>
>
>
>
>
> On Tue, 21 Jan 2020 at 18:06, Guenter Fritz <
> guenter.fritz.phenix.c...@gmail.com> wrote:
>
> Dear Chris, are there any metal ions in your buffer or in your protein. We
> had a similar looking case. A Zn2+ ion bridged two monomers. Our protein is
> a Zn2+ binding protein. The Zn2+ originated from some denatured protein in
> the drop. No extra Zn2+ was in the crystallization buffer.
>
>
>
> http://www.rcsb.org/structure/5CHT
>
> https://www.nature.com/articles/nsmb.3371
>
> HTH
>
> Guenter
>
> Dear CCP4BB Users,
>
>
>
> I've recently solved the ~2.2 angstrom structure of a protein. In my
> electron density there are unusual monomer-monomer interfaces involving
> pairs of His and Cys residues (see https://ibb.co/wdWBcdk). Note the
> positive Fo-Fc density between the four side chains. As there is not
> adequate space for a water molecule or metal ion, perhaps the Cys residues
> are partially tied up disulfide bonds? However, the protein looks to be
> fully monomeric based on LC-MS measurements. Has anyone else observed
> crystal-driven formation of disulfide bridges?
>
>
>
> Aside from this region, there is no extensive interface between momoners,
> and PDBePISA suggests a monomeric state

Re: [ccp4bb] Unusual monomer-monomer interface in crystal

[Chris Fage]

Thanks to Guenter and Eleanor for their replies. I mentioned that there is
not adequate space for a metal ion at the described interfaces.
Nevertheless, placement of a metal ion, followed by refinement in Phenix,
repositions the side chains significantly so as to make room for the ion
without distorting geometry. There is also a very strong difference signal
centered between the four side chains. This agrees with the MS data, which
indicate a monomeric state without a disulfide linkage. Now, I just need to
identify the metal. A Zn2+ seems to fit well based on coordination number
and interatomic distances, as Guenter exemplified.

Best wishes,
Chris

On Tue, Jan 21, 2020 at 6:33 PM Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> Easy to check whether it is a metal by looking at an anomalous difference
> map..
> But there are examples of di-sulphides formed between symmetry related
> molecules..
> Query the wwwpdb -
>
>
> On Tue, 21 Jan 2020 at 18:06, Guenter Fritz <
> guenter.fritz.phenix.c...@gmail.com> wrote:
>
>> Dear Chris, are there any metal ions in your buffer or in your protein.
>> We had a similar looking case. A Zn2+ ion bridged two monomers. Our protein
>> is a Zn2+ binding protein. The Zn2+ originated from some denatured protein
>> in the drop. No extra Zn2+ was in the crystallization buffer.
>>
>> http://www.rcsb.org/structure/5CHT
>> https://www.nature.com/articles/nsmb.3371
>> HTH
>> Guenter
>>
>> Dear CCP4BB Users,
>>
>> I've recently solved the ~2.2 angstrom structure of a protein. In my
>> electron density there are unusual monomer-monomer interfaces involving
>> pairs of His and Cys residues (see https://ibb.co/wdWBcdk). Note the
>> positive Fo-Fc density between the four side chains. As there is not
>> adequate space for a water molecule or metal ion, perhaps the Cys residues
>> are partially tied up disulfide bonds? However, the protein looks to be
>> fully monomeric based on LC-MS measurements. Has anyone else observed
>> crystal-driven formation of disulfide bridges?
>>
>> Aside from this region, there is no extensive interface between momoners,
>> and PDBePISA suggests a monomeric state.
>>
>> Thanks in advance for any advice!
>>
>> Best wishes,
>> Chris
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>>
>>
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[ccp4bb] Unusual monomer-monomer interface in crystal

[Chris Fage]

Dear CCP4BB Users,

I've recently solved the ~2.2 angstrom structure of a protein. In my
electron density there are unusual monomer-monomer interfaces involving
pairs of His and Cys residues (see https://ibb.co/wdWBcdk). Note the
positive Fo-Fc density between the four side chains. As there is not
adequate space for a water molecule or metal ion, perhaps the Cys residues
are partially tied up disulfide bonds? However, the protein looks to be
fully monomeric based on LC-MS measurements. Has anyone else observed
crystal-driven formation of disulfide bridges?

Aside from this region, there is no extensive interface between momoners,
and PDBePISA suggests a monomeric state.

Thanks in advance for any advice!

Best wishes,
Chris



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Re: [ccp4bb] Water at AU boundary

[Chris Fage]

Thanks to Eugene, Mark, and Rajiv. All suggested a change in occupancy.

Best wishes,
Chris

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On Wed, Nov 6, 2019 at 10:26 AM Eugene Osipov  wrote:

> Chris,
> you can reduce occupancy of the water molecule, like: 0.5 if water lies on
> 2-fold axis, or 1/3 if it is on 3-fold axis and so on
>
> ср, 6 нояб. 2019 г. в 11:18, Chris Fage :
>
>> Dear BB Users,
>>
>> Within my unit cell, I see overlapping spherical blobs of Fo-Fc and
>> 2Fo-Fc density in Coot. These blobs appear directly at the symmetry
>> boundary, such that building a water molecule at the position results in
>> overlaid symmetry-related molecules (see water 420, between three copies of
>> His253). Is there a way to add this water without suffering from symmetry
>> clashes, or should the density be ignored? Thanks in advance for your
>> suggestions.
>>
>> Image: https://ibb.co/HYFFcbt
>>
>> Best wishes,
>> Chris
>>
>>
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>>
>> --
>>
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>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>
> --
> Evgenii Osipov
> Laboratory for Biocrystallography,
> Department of Pharmaceutical Sciences,
> KU Leuven O
>



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[ccp4bb] Water at AU boundary

[Chris Fage]

Dear BB Users,

Within my unit cell, I see overlapping spherical blobs of Fo-Fc and 2Fo-Fc
density in Coot. These blobs appear directly at the symmetry boundary, such
that building a water molecule at the position results in overlaid
symmetry-related molecules (see water 420, between three copies of His253).
Is there a way to add this water without suffering from symmetry clashes,
or should the density be ignored? Thanks in advance for your suggestions.

Image: https://ibb.co/HYFFcbt

Best wishes,
Chris


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Re: [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Chris Fage]

Dear Murpholino,

Yes, I think you're right. For now, directly exporting my maps from Coot is
the most efficient solution. The normalization commands will certainly be
helpful to me and other users in the future, though. Thanks!

Best,
Chris

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On Mon, Sep 30, 2019 at 9:02 PM Murpholino Peligro 
wrote:

> I think this is part of the problem.
> https://pymolwiki.org/index.php/Normalize_ccp4_maps
>
> El lun., 30 de sep. de 2019 a la(s) 10:09, Chris Fage (fage...@gmail.com)
> escribió:
>
>> Hi Paul,
>>
>> After exporting the maps from Coot, they are directly comparable in
>> Pymol. That's quite a convenient feature I was never aware of. Thanks!
>>
>> Yes, I was comparing the map from FFT in Coot and PyMOL.
>>
>> Best,
>> Chris
>>
>>
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>>
>> On Mon, Sep 30, 2019 at 3:04 PM Paul Emsley 
>> wrote:
>>
>>> On 30/09/2019 13:00, Chris Fage wrote:
>>> > Dear Paul, Herman, Robbie, and Santosh,
>>> >
>>> >
>>> > My version of Pymol doesn't support loading of mtz files. I think it's
>>> > only in the incentive version.
>>> >
>>> > Paul wrote: "No need to do this - just export the map (or the map
>>> > fragment)." I'm not sure how to do this without going through FFT!
>>>
>>>
>>> File -> Export Map...
>>>
>>>
>>> >
>>> > But when I generate a new set of maps with F1=FWT and PHI=PHWT and
>>> > load them into Pymol, they are still not comparable to those in Coot.
>>> > Again, the maps for ligands 1 and 2 in Pymol look about the same.
>>>
>>>
>>> Are you comparing the map from FFT in Coot and PyMOL?
>>>
>>>
>>> Paul.
>>>
>>> 
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
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>>
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Re: [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Chris Fage]

Hi Paul,

After exporting the maps from Coot, they are directly comparable in Pymol.
That's quite a convenient feature I was never aware of. Thanks!

Yes, I was comparing the map from FFT in Coot and PyMOL.

Best,
Chris

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On Mon, Sep 30, 2019 at 3:04 PM Paul Emsley 
wrote:

> On 30/09/2019 13:00, Chris Fage wrote:
> > Dear Paul, Herman, Robbie, and Santosh,
> >
> >
> > My version of Pymol doesn't support loading of mtz files. I think it's
> > only in the incentive version.
> >
> > Paul wrote: "No need to do this - just export the map (or the map
> > fragment)." I'm not sure how to do this without going through FFT!
>
>
> File -> Export Map...
>
>
> >
> > But when I generate a new set of maps with F1=FWT and PHI=PHWT and
> > load them into Pymol, they are still not comparable to those in Coot.
> > Again, the maps for ligands 1 and 2 in Pymol look about the same.
>
>
> Are you comparing the map from FFT in Coot and PyMOL?
>
>
> Paul.
>
> 
>
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Re: [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Chris Fage]

Hi Robbie,

I guess that solves part of the problem. But when I generate a new set of
maps with F1=FWT and PHI=PHWT and load them into Pymol, they are still not
comparable to those in Coot. Again, the maps for ligands 1 and 2 in Pymol
look about the same.

Best,
Chris

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On Mon, Sep 30, 2019 at 1:26 PM Robbie Joosten 
wrote:

> Hi Chris,
>
> You made an Fobs map not a 2Fo-Fc map. You can leave sigma empty if you
> want to make a map in this case.
>
> Cheers,
> Robbie
>
> > -Original Message-
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> > Chris Fage
> > Sent: Monday, September 30, 2019 14:00
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol
> >
> > Dear Paul, Herman, Robbie, and Santosh,
> >
> > Thanks for your quick replies.
> >
> > The FFT-generated maps and mtz maps look roughly equivalent in Coot, but
> > there are minor differences even at the same e/A^3 level (the mtz maps
> > actually look a bit weaker). I generated them using the default settings
> in
> > FFT: simple map, format to cover asymmetric unit, F1=F_XDSdataset,
> > Sigma=SIGF_XDSdataset, PHI=PHIC. Perhaps, as Robbie mentioned, the
> > problem lies in column selection, since Coot uses FWT and PHWT. I can
> assign
> > F1 and PHI to these values, respectively, but what should I choose for
> Sigma?
> >
> > My version of Pymol doesn't support loading of mtz files. I think it's
> only in
> > the incentive version.
> >
> > Paul wrote: "No need to do this - just export the map (or the map
> > fragment)." I'm not sure how to do this without going through FFT!
> >
> > I can also try generating maps in Phenix, as Santosh suggested. However,
> if
> > the maps can be directly exported, as Paul suggested, I would prefer to
> > follow that route.
> >
> > Best wishes,
> > Chris
> >
> >
> >
> >
> >  <http://www.avg.com/email-
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> >
> >
> > On Mon, Sep 30, 2019 at 12:49 PM Santhosh Gatreddi
> >  wrote:
> >
> >
> >   Hi Chris,
> >
> >   I also observed similar thing when I have generated 2Fo-Fc maps in
> > CCP4 FFT. But when I have generated 2Fo-Fc maps in Phenix then my maps
> > were similar  in Pymol and Coot. I request you to generate maps with
> Phenix
> > and verify it in Pymol. It worked for me.
> >
> >   One possible reason for this is map averaging in Pymol. Make sure
> > that you uncheck it before loading your map in Pymol.
> >
> >   I hope that above suggestion works for you.
> >
> >   Regards,
> >   Santhosh
> >
> >   On Mon, Sep 30, 2019 at 4:06 PM Chris Fage 
> > wrote:
> >
> >
> >   Dear All,
> >
> >   I recently obtained structures of a protein bound to two
> > different small molecules. When viewing the structures in Coot with a
> similar
> > contour setting, the 2Fo-Fc map around ligand 1 is clearly much weaker
> than
> > that around ligand 2.However, after generating 2Fo-Fc maps in FFT and
> > loading them in Pymol (again, choosing equal contour levels), the maps
> > surrounding ligands 1 and 2 have nearly the same quality. Is there a
> > difference in scaling between the two programs that can account for this?
> > Thanks for any advice!
> >
> >   Best wishes,
> >   Chris
> >
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> > 
> >
> >
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> >
> >
> >
> >   --
> >
> >   With regards
> >
> >  

Re: [ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Chris Fage]

Dear Paul, Herman, Robbie, and Santosh,

Thanks for your quick replies.

The FFT-generated maps and mtz maps look roughly equivalent in Coot, but
there are minor differences even at the same e/A^3 level (the mtz maps
actually look a bit weaker). I generated them using the default settings in
FFT: simple map, format to cover asymmetric unit, F1=F_XDSdataset,
Sigma=SIGF_XDSdataset, PHI=PHIC. Perhaps, as Robbie mentioned, the problem
lies in column selection, since Coot uses FWT and PHWT. I can assign F1 and
PHI to these values, respectively, but what should I choose for Sigma?

My version of Pymol doesn't support loading of mtz files. I think it's only
in the incentive version.

Paul wrote: "No need to do this - just export the map (or the map
fragment)." I'm not sure how to do this without going through FFT!

I can also try generating maps in Phenix, as Santosh suggested. However, if
the maps can be directly exported, as Paul suggested, I would prefer to
follow that route.

Best wishes,
Chris




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On Mon, Sep 30, 2019 at 12:49 PM Santhosh Gatreddi 
wrote:

> Hi Chris,
>
> I also observed similar thing when I have generated 2Fo-Fc maps in CCP4
> FFT. But when I have generated 2Fo-Fc maps in Phenix then my maps were
> similar  in Pymol and Coot. I request you to generate maps with Phenix and
> verify it in Pymol. It worked for me.
>
> One possible reason for this is map averaging in Pymol. Make sure that you
> uncheck it before loading your map in Pymol.
>
> I hope that above suggestion works for you.
>
> Regards,
> Santhosh
>
> On Mon, Sep 30, 2019 at 4:06 PM Chris Fage  wrote:
>
>> Dear All,
>>
>> I recently obtained structures of a protein bound to two different small
>> molecules. When viewing the structures in Coot with a similar contour
>> setting, the 2Fo-Fc map around ligand 1 is clearly much weaker than that
>> around ligand 2.However, after generating 2Fo-Fc maps in FFT and loading
>> them in Pymol (again, choosing equal contour levels), the maps surrounding
>> ligands 1 and 2 have nearly the same quality. Is there a difference in
>> scaling between the two programs that can account for this? Thanks for any
>> advice!
>>
>> Best wishes,
>> Chris
>>
>>
>> <http://www.avg.com/email-signature?utm_medium=email_source=link_campaign=sig-email_content=webmail>
>>  Virus-free.
>> www.avg.com
>> <http://www.avg.com/email-signature?utm_medium=email_source=link_campaign=sig-email_content=webmail>
>> <#m_-501199200969567658_m_-8793236030949751887_DAB4FAD8-2DD7-40BB-A1B8-4E2AA1F9FDF2>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>
> --
>
>
>
>
>
>
>
>
>
>
>
> *With regardsSanthosh Gatreddi (Research Scholar)c/o Dr.Insaf Ahmed
> Qureshi, Dept. of Biotechnology & Bioinformatics,School of
> lifesciences,University of
> Hyderabad,Hyderabad-500046(A.P),India.Ph.no-9160628684.*
>



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[ccp4bb] 2Fo-Fc maps in Coot vs. Pymol

[Chris Fage]

Dear All,

I recently obtained structures of a protein bound to two different small
molecules. When viewing the structures in Coot with a similar contour
setting, the 2Fo-Fc map around ligand 1 is clearly much weaker than that
around ligand 2.However, after generating 2Fo-Fc maps in FFT and loading
them in Pymol (again, choosing equal contour levels), the maps surrounding
ligands 1 and 2 have nearly the same quality. Is there a difference in
scaling between the two programs that can account for this? Thanks for any
advice!

Best wishes,
Chris


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[ccp4bb] OT: HELIX and SHEET records not read by Pymol

[Chris Fage]

Dear All,

Pymol seems to ignore the HELIX and SHEET records in my coordinate file,
and instead assigns secondary structural features according to the default
method. Unless my understanding is flawed, these records should override
Pymol's assignment...?

I generated the records with the Stride Web Interface and converted them to
PDB format with Emma Rath's website (
http://www.canoz.com/sdh/STRIDEtoPDBsecondarystruct.pl).

Does anyone have any ideas? Thanks!

Best wishes,
Chris


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Re: [ccp4bb] coot: obtaining a clickable list of outiers in a Ramachandran plot

[Chris Fage]

Hi Laurent,

I seem to recall being able to click on outliers in the output of the
phenix.refine GUI, which links to a Coot window. This would, however,
require refinement in the Phenix suite rather than the CCP4 one.

Best,
Chris

On Thu, Mar 22, 2018 at 10:27 AM, maveyrau 
wrote:

> Hi all,
>
> I’m currently refining quite a big oligomer (about 3500 residues) using
> refmac/coot, at a 2.85 A resolution. The Ramachandran tool in Coot
> indicates  about 100 outliers, and I would like to check them individually…
> Is there a way to get a clickable list of outliers? I know I can click on
> the Ramachandran plot directly, but with so many residues it’s quite
> impossible to go through all of them…
>
> Thanks for any advices…
> Laurent
> --
> Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr
> P I C T   ---  Plateforme Intégrée de Criblage de Toulouse
> Université  Paul  Sabatier /  CNRS  /  I.P.B.S.  UMR  5089
> Département BiologieStructurale   et   Biophysique
> http://cribligand.ipbs.fr   http://www.ipbs.fr
> 205 route de Narbonne  31077 TOULOUSE Cedex FRANCE
> Tél: +33 (0)561 175 435   Mob.: +33 (0)646 042 111
> --
>
>
>
>
>

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Chris Fage]

Dear All,

Thank you for the many suggestions. After sending my first message to the
BB, I tried exchanging the sample into buffer containing 5 mM EDTA and also
into buffer at pH 9.0 (using BICINE). Neither of these appear to have
helped the instability--precipitation still occurred within ~1 min of
removal of the tube from ice. The theoretical pI is ~6.1, which is far from
my working pH, although as Mark indicated the calculated pI may be
inaccurate, and I may even need to try a more acidic pH. I will test the
ideas provided by everyone over the next week and leave some feedback. It
may be that I need to prepare a different truncation, as this domain is
excised from a larger covalent assembly. I have purified homologs trimmed
at a similar position in the past, but of course that doesn't guarantee
good behavior in my current system.

Best,
Chris

On Fri, Jul 14, 2017 at 10:20 AM, Eugene Osipov <e.m.osi...@gmail.com>
wrote:

>  Hi, try to add 1-5 mM EDTA, because trace amounts of metals cause
> precipitation of tagged proteins.
>
> 14 июля 2017 г. 1:40 пользователь "Chris Fage" <fage...@gmail.com>
> написал:
>
> Dear CCP4BB Community,
>>
>> This week, I purified a nicely overexpressing protein by Ni-NTA followed
>> by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
>> fractions to ~1 mL, transferred the spin filter to ice, and then collected
>> 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
>> heavily in the pipet tip before I could dispense it onto the Nanodrop
>> pedestal, directly adjacent to my ice box. This effect seems to be abated
>> at 4 C, as the protein remained stable in cold room-chilled pipet tips.
>> However, the protein also precipitated heavily when overnight at 4 C in 1
>> mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
>> overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
>> 10% glycerol) prior to gel filtration. Has anyone experienced and resolved
>> a similar issue before? Do any useful additives come to mind?
>>
>> Things I have tried with the gel filtration sample:
>> -Exchanging buffer to restore the salt concentration to Ni-NTA levels
>> (e.g. 500 mM).
>> -Exchanging buffer to add 10% glycerol.
>> -Simply diluting the protein in gel filtration buffer to rule out
>> concentration dependence.
>>
>> In each case, the protein precipitates to a milky solution within about a
>> minute of removal from ice (I am working with 20-50 uL volumes in PCR
>> tubes).
>>
>> Many thanks for any suggestions!
>>
>> Best,
>> Chris
>>
>

[ccp4bb] Protein rapidly precipitates when off ice

[Chris Fage]

Dear CCP4BB Community,

This week, I purified a nicely overexpressing protein by Ni-NTA followed by
gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
fractions to ~1 mL, transferred the spin filter to ice, and then collected
2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
heavily in the pipet tip before I could dispense it onto the Nanodrop
pedestal, directly adjacent to my ice box. This effect seems to be abated
at 4 C, as the protein remained stable in cold room-chilled pipet tips.
However, the protein also precipitated heavily when overnight at 4 C in 1
mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
10% glycerol) prior to gel filtration. Has anyone experienced and resolved
a similar issue before? Do any useful additives come to mind?

Things I have tried with the gel filtration sample:
-Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g.
500 mM).
-Exchanging buffer to add 10% glycerol.
-Simply diluting the protein in gel filtration buffer to rule out
concentration dependence.

In each case, the protein precipitates to a milky solution within about a
minute of removal from ice (I am working with 20-50 uL volumes in PCR
tubes).

Many thanks for any suggestions!

Best,
Chris

Re: [ccp4bb] Shape similarity of ligand binding sites

[Chris Fage]

Hi Stephen,

I've had some luck calculating ligand-binding cavity volume with the 3v
website (http://3vee.molmovdb.org/). You might want to give it a shot.

Best,
Chris

On Wed, Jun 14, 2017 at 12:42 PM,  <
stephen.c...@rc-harwell.ac.uk> wrote:

> Dear ccp4bb,
>
> I am trying to compare the shape of a ligand binding site in my protein
> with that of some homologues and mutants and was wondering how others go
> about this?  I specifically want to compare the shapes of the surface
> (similar to an sc analysis of an interface) rather than the positions of
> the atoms in the residues that make up the site.  I have come across
> PESDserv, but this doesn't do quite what I would like.  Any suggestions
> would be very welcome.
>
> Best wishes,
>
> Steve
>
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
>
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Re: [ccp4bb] structure determination from a hollow crystal

[Chris Fage]

Hi Eike,

I wouldn't necessarily let the morphology deter you. While working on my
PhD, a colleague collected beautiful data from hollow crystals.

If the crystals collapse while looping, is it possible for you to perform
"micro-surgery" on the crystal so that you only have a single face?
Sometimes you can obtain very nice datasets from plates.

Best,
Chris

On Wed, Nov 30, 2016 at 8:20 AM, Schulz, Eike-Christian <
eike.sch...@mpsd.mpg.de> wrote:

> Dear all,
>
>
>
> Is there a documented case of a structure determination from a hollow
> crystal? We have the unfortunate situation that a protein only crystallizes
> as a hollow tube, where the inside is filled with solvent, which ruins the
> diffraction patterns.
>
>
>
> I was often confronted with similar situations, but in the previous cases
> I could always find a suitable crystal (part) or a different
> crystallization condition saved me.
>
>
>
> If anybody had general advise on how to proceed or could point me to
> relevant literature it would be much appreciated.
>
>
>
> With best regards
>
>
>
> Eike
>
>
>
>

Re: [ccp4bb] Why Does Detwinning Not Work?

[Chris Fage]

Sorry--I think you were referring to phasing, not refinement. I hope my
message is still relevant.

Cheers,
Chris

On Tue, Oct 11, 2016 at 10:39 AM, Chris Fage <cdf...@gmail.com> wrote:

> Dear Jacob,
>
> I'm not an expert on the topic, but from my experiences with twinning I
> can agree with you. I recently solved my second twinned structure by MR
> (twin fraction of 0.43, as estimated by Xtriage). Performing twin
> refinement in Refmac or phenix.refine dropped the R-factors, as expected,
> but worsened the geometry considerably without a noticeable improvement in
> the maps. For this reason, I opted *not* to go with the twin refinement...
> I don't know if others would make the same choice, though it seemed
> reasonable to me. Besides, my Rwork/Rfree is down to 0.25/0.29, which ain't
> too shabby for 2.6 A resolution.
>
> Cheers,
> Chris
>
> On Tue, Oct 11, 2016 at 2:15 AM, Keller, Jacob <kell...@janelia.hhmi.org>
> wrote:
>
>> Dear Crystallographers,
>>
>>
>>
>> Based on some data sets I have looked at and anecdotal-type evidence here
>> and there I have gotten the impression that detwinning does not help in
>> structure solution. (Please let me know if you have a case where detwinning
>> saved the day.) Is there a clear answer to this enigma anywhere, to
>> anyone’s knowledge? Wouldn’t it seem that **any** detwinning would be
>> better than **no** detwinning? I understand that the errors explode as
>> one approaches 50% twins and does detwinning, but still, I don’t think one *
>> *loses** information by detwinning, right? Take the case of a 33% twin:
>> since the twin-reflections are on average about half the intensity of the
>> non-twin, and since they are generally not correlated in intensity, isn’t
>> this like having noise added at 50% of the measured intensity? So why does
>> detwinning make things worse generally? Is there something wrong in the
>> assumptions underlying the detwinning algorithm, or perhaps something about
>> the calculation that throws things off?
>>
>>
>>
>> A related sub-enigma: why is MR generally immune to twinning, but
>> anomalous methods are susceptible?
>>
>>
>>
>> All the best,
>>
>>
>>
>> Jacob Keller
>>
>>
>>
>> ***
>>
>> Jacob Pearson Keller, PhD
>>
>> Research Scientist
>>
>> HHMI Janelia Research Campus / Looger lab
>>
>> Phone: (571)209-4000 x3159
>>
>> Email: kell...@janelia.hhmi.org
>>
>> ***
>>
>>
>>
>
>

Re: [ccp4bb] Why Does Detwinning Not Work?

[Chris Fage]

Dear Jacob,

I'm not an expert on the topic, but from my experiences with twinning I can
agree with you. I recently solved my second twinned structure by MR (twin
fraction of 0.43, as estimated by Xtriage). Performing twin refinement in
Refmac or phenix.refine dropped the R-factors, as expected, but worsened
the geometry considerably without a noticeable improvement in the maps. For
this reason, I opted *not* to go with the twin refinement... I don't know
if others would make the same choice, though it seemed reasonable to me.
Besides, my Rwork/Rfree is down to 0.25/0.29, which ain't too shabby for
2.6 A resolution.

Cheers,
Chris

On Tue, Oct 11, 2016 at 2:15 AM, Keller, Jacob 
wrote:

> Dear Crystallographers,
>
>
>
> Based on some data sets I have looked at and anecdotal-type evidence here
> and there I have gotten the impression that detwinning does not help in
> structure solution. (Please let me know if you have a case where detwinning
> saved the day.) Is there a clear answer to this enigma anywhere, to
> anyone’s knowledge? Wouldn’t it seem that **any** detwinning would be
> better than **no** detwinning? I understand that the errors explode as
> one approaches 50% twins and does detwinning, but still, I don’t think one *
> *loses** information by detwinning, right? Take the case of a 33% twin:
> since the twin-reflections are on average about half the intensity of the
> non-twin, and since they are generally not correlated in intensity, isn’t
> this like having noise added at 50% of the measured intensity? So why does
> detwinning make things worse generally? Is there something wrong in the
> assumptions underlying the detwinning algorithm, or perhaps something about
> the calculation that throws things off?
>
>
>
> A related sub-enigma: why is MR generally immune to twinning, but
> anomalous methods are susceptible?
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
> ***
>
> Jacob Pearson Keller, PhD
>
> Research Scientist
>
> HHMI Janelia Research Campus / Looger lab
>
> Phone: (571)209-4000 x3159
>
> Email: kell...@janelia.hhmi.org
>
> ***
>
>
>

Re: [ccp4bb] CrystalClear tape rolls

[Chris Fage]

Hi Mark,

I recently ordered HD Clear Sealing Tape, 3 inches wide from Jena:
http://www.jenabioscience.com/cms/en/1/catalog/287_sealing_tape.html

Best,
Chris

On Mon, Dec 1, 2014 at 12:18 PM, Mark J van Raaij mjvanra...@cnb.csic.es
wrote:

 you are right, I must have been looking with my ears when I checked this
 morning.
 https://hamptonresearch.com/product_detail.aspx?cid=10sid=88pid=271

 Mark J van Raaij
 Lab 20B
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij







 On 1 Dec 2014, at 12:05, Andreas Förster wrote:

  Hi Mark,
 
  last time I checked (few months back), Hampton still sold tape. Shipping
 to Europe is extortionate but if you combine with screens and other
 goodies, it's just about bearable.
 
 
  Andreas
 
 
 
  On 01/12/2014 10:24, Mark J van Raaij wrote:
  Dear All,
 
  Just wondering what the current situation on CrystalClear tray sealing
 tape is.
  Molecular Dimensions, Hampton, Jena seem to be selling mainly sheets or
 strips precut for plates - I guess related with the fact that the consumer
 box sealing tape changed specifications and now clouds over with certain
 reservoir solutions. See:
  https://www.mail-archive.com/ccp4bb@dl.ac.uk/msg00622.html
  We are stilll using rolls which came with plates we bought years back,
 but now only have two rolls left - so I am wondering if there is a more
 economic solution than buying sheets or strips.
 
  Greetings,
 
  Mark
 
  Mark J van Raaij
  Lab 20B
  Dpto de Estructura de Macromoleculas
  Centro Nacional de Biotecnologia - CSIC
  c/Darwin 3
  E-28049 Madrid, Spain
  tel. (+34) 91 585 4616
  http://www.cnb.csic.es/~mjvanraaij
 
 
  --
   Andreas Förster
X-ray Crystallography Facility Manager
Centre for Structural Biology
   Imperial College London

Re: [ccp4bb] REFMAC - TWIN OPTION

[Chris Fage]

Hi Bernie,

I agree with Herman. I think you will be all set if you simply change the no 
twin refinement option near the top of the Refmac interface to intensity 
based or amplitude based twin refinement. Refmac will determine the 
appropriate twin operators/fractions and refine. 

Best,
Chris 

 On Sep 17, 2014, at 14:19, Santarsiero, Bernard D. b...@uic.edu wrote:
 
 Can someone point me to bulletpoint documentation on using the twin
 refinement in CCP4?
 
 Here's what I did.
 
 1.  I'm in space group P3, and the see a very clean diffraction pattern
 that looks like one single lattice. Very clean spots, so merohedral
 twinning.
 
 2.  You can use various programs to estimate the twin fraction and select
 various twin laws.
 
 3.  You run DETWIN in the UTILITIES section of DATA REDUCTION and
 ANALYSIS. I have both intensities IMEAN and amplitudes FMEAN, and selected
 amplitudes. The FMEAN changes to something like FMEAN_detw. You have to
 select a twin fraction, but does it matter what the twin factor you assign
 is?  (It seems to get refined, or just estimated, in refinement?)
 
 4.  There are potentially 3 twin laws, so do you run DETWIN three times? 
 You can, in fact, get amplitudes that are FMEAN_detw_detw_detw.  I've run
 it once and three times, and seems to give the same result in REFMAC
 refinement.
 
 5.  Select the Free R subset. Will uniqueify choose correctly?
 
 6.  In REFMAC, it looks for the F_detw amplitude, or you can select it.
 Then it seems to select and test three twin laws, even if you only ran
 DETWIN once, with one selected twin law. It tests if the twin laws give
 low R(merge).  If not, it tosses them out. It seems like the R(merge)
 calculation depends on the starting model, and not just on the modified
 Fobs data. It also seems like it tries to calculate the twin fraction, and
 then refine the structure. Or, is it just giving an estimate, and I need
 to go back to original data and rerun DETWIN?
 
 7.  I tried to work with intensities instead of amplitudes, and it can
 blow up. I've also refined, got a good refinement (3 twin laws, each with
 twin fraction around 0.1, Rcryst = 19%, Rfree = 21%), rebuilt one chain of
 the structure, and it blows up, suggesting no twinning, but Rs around 35%.
 
 Does REFMAC refine the twin fraction?
 Do I have to assign a twin fraction in DETWIN, or does it just set up the
 relationships between reflections and amplitudes?
 Should I try to work with intensities or amplitudes.  The calculation is
 presumably done on intensities, but seems to be more unstable using that
 option.
 
 I haven't tried the PHENIX option. Just a quick step-by-step guide on what
 to do would be useful.
 
 Thanks,
 
 Bernie
 -- 
 Bernard D. Santarsiero
 Research Professor
 Center for Structural Biology
 University of Illinois at Chicago
 MC870  3070MBRB  900 South Ashland Avenue
 Chicago, IL 60607-7173  USA
 (312) 413-0339 (office)
 (312) 413-9303 (FAX)
 http://www.uic.edu/labs/bds
 http://scholar.google.com/citations?user=fGauLBMJ

[ccp4bb]

[Chris Fage]

Hi Prashant,

I typically stop the centrifuge once in awhile and pipet up/down to prevent
the sample from over-concentrating. Depending on how sensitive the sample
is, you may want to do this once every 10-60 min.

Hope this helps,
Chris


On Tue, Aug 19, 2014 at 1:42 PM, Prashant Deshmukh 
prashantbiophys...@gmail.com wrote:

 Hi,
 i am concentrating my protein using centricon filter, but it is
 precipitated soon. Please help me solving this problem.
 Thanks.
 Prashant Deshmukh
 Dept. of Biophysics,
 NIMHANS,
 Bangalore 560 029,
 E-mail:prashantbiophys...@gmail.com
 Mob.No.: +919620986525

Re: [ccp4bb] Heavy Atom Phasing

[Chris Fage]

Hi Rhys,

Have you tried quick back-soaks into solutions lacking the heavy atoms? This 
can reduce radiation decay caused by absorption of X-rays by overabundant heavy 
atoms. 

Best, 
Chris

 On Jul 27, 2014, at 4:48 PM, RHYS GRINTER r.grinte...@research.gla.ac.uk 
 wrote:
 
 Hi All,
 
 I thought I might put a question to the community, with the hope of getting 
 some tips of the best way to proceed with my heavy atom phasing problem.
 I'm working on solving the structure of an integral beta-barrel membrane 
 protein of approximately 100 kDa. I've crystallised protein, growing some 
 very flimsy needle like crystals, and collected datasets to around 3.1 A.
 I then produced selenomet derivative protein and repeated crystallisation 
 trials in the same conditions and also repeated broad screens, however the 
 derivative protein failed to produce crystals that diffracted beyond 10 A (in 
 fact it barely crystallises at all).
 So I've moved on to heavy atom soaks and have had some success with 
 tetrachloroplatinate and tetranitroplatinate compounds, in that the crystals 
 didn't dissolve (as they did with gold and samarium compounds) and diffracted 
 to some degree. I collected SAD data to around 6.5 A from these crystals and 
 there seems to be anomolous signal. However, while I get a good CC of 0.4 
 from HYSS in phenix with this dataset and the phaser EP FOM is 0.56, the maps 
 before and after DM are uninterpretable. I'm guessing the quality and 
 resolution of the data I collected just aren't good enough (the data is 
 reasonably anisotropic).
 I performed the metal soaking, by taking a small amount of the platinate salt 
 and adding it to the crystallisation drop as the crystals are extremely 
 fragile and don't stand up well to handling through a soaking or cryo 
 solution. Leaving the crystals to soak for 48 hours and then, freezing them 
 directly. The solution is on the border of cryoprotection (the conditions has 
 PEG2000MME and PVP and the precipitants), but with native crystals this 
 doesn't seem to be a parameter which affects diffraction. The crystals are 
 very variable in performance, so while I feel that the heavy atom soaking has 
 compromised their diffractability to a degree, inherent variation may play a 
 part.
 
 What I was wondering is if some one with more experience than me found 
 themselves in this position, how would they proceed? Questions which spring 
 to mind are, how much heavy atom compound do people add and how long do they 
 soak for? Is there anyway I can squeeze something out of the anomalous data I 
 have, given I have 'reasonable' native data, or will poor quality data give 
 spuriously positive statistics for heavy atom phasing? And are there any 
 tricks people have experienced to improve performance of crystals like these 
 (aside from the usual seeding, additives, different detergents etc which I 
 have spend a fair bit of time on optimization already).
 
 Thanks in advance,
 
 Rhys 

Re: [ccp4bb] Salt!

[Chris Fage]

Hi Catherine,

If they are indeed salt crystals, have you tried preparing different
truncations of the gene encoding your target? I've seen a number of
successes in which, after a codon or two was added to the termini of the
gene being overexpressed, the target crystallized beautifully.

Best,
Chris


On Wed, Jul 16, 2014 at 9:55 AM, Bishop, Catherine E. cati...@ou.edu
wrote:

  I have been attempting to obtain a protein crystal of my protein for
 just over 2 years at this point.  We have attempted removing the tag,
 binding the protein to its ligand, removing as much salt as possible
 (crashes at at too low a salt concentration)--this lead us to try reverse
 vapor diffusion--seeding, additive trays, optimization around the
 conditions an ortholog of one of the domains crystallized well in, and a
 plethora of other methods. We do get crystals, but they all diffract as
 salt. Most of these salts are found in wells containing a cation (ie.
 lithium sulfate, nickel chloride, magnesium acetate, cobalt chloride,
 etc.). When crystals are too small to shoot, I do set up optimized trays;
 however, if I get larger crystals, they diffract as salt as well. Most of
 these set ups, at this point, have also been conducted in hands other than
 mine and at other facilities known for their successful crystallization of
 proteins.

 Has anyone else run into this problem and seen light at the other side??
 Any suggestions are appreciated.

Re: [ccp4bb] How to store PEG screens

[Chris Fage]

Hi Jerome,

-I have heard that PEG solutions can become unstable in light. We usually
store our block in the fridge, where photons are scant anyway. For any
stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not
sure about freezing them.
-Some labs (not ours) evidently prepare buffered stocks of PEG solutions,
as their pHs tend to shift with time. This is something I've been meaning
to try. Of course, you may need to worry about buffer components that are
incompatible with crystal hits.

Hope this helps,
Chris


On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu
wrote:

 Dear all,
 I have 3 short questions about PEG solutions:
 Does anyone know the best way to store crystallization screening blocks
 that contain PEG 3350?
 Is it a good idea to freeze the PEG solutions at -80°C and thaw them
 before use?
 Would the freeze-thaw process considerably alter the PEG chain lengths?

 Thank you,
 -Jerome

 Jerome Nwachukwu

Re: [ccp4bb] How to store PEG screens

[Chris Fage]

I also used to store my PEG solutions in light, and my stocks do sit
out on the bench. I can't say for sure whether light or temperature
make a difference, but I like to heed what seem like superstitions in
crystallography to eliminate variables. We purchase our screens from
Qiagen, who suggests that blocks be stored in the fridge and warmed to
RT before use.

Best,
Chris

On 7/14/14, Nicholas Larsen nicholas_lar...@h3biomedicine.com wrote:
 I don't think storage matters.  I doubt Hampton stores their PEG stock
 solutions at -80 before they ship out to customers.
 I've solved tons of structures leaving my PEGS and PEG screens at RT in the
 light.

 Nick


 On Mon, Jul 14, 2014 at 12:32 PM, Chris Fage cdf...@gmail.com wrote:

 Hi Jerome,

 -I have heard that PEG solutions can become unstable in light. We usually
 store our block in the fridge, where photons are scant anyway. For any
 stocks that I prepare, I wrap the tube/bottle in aluminum foil. I'm not
 sure about freezing them.
 -Some labs (not ours) evidently prepare buffered stocks of PEG solutions,
 as their pHs tend to shift with time. This is something I've been meaning
 to try. Of course, you may need to worry about buffer components that are
 incompatible with crystal hits.

 Hope this helps,
 Chris


 On Mon, Jul 14, 2014 at 9:33 AM, Jerome Nwachukwu jnwac...@scripps.edu
 wrote:

 Dear all,
 I have 3 short questions about PEG solutions:
 Does anyone know the best way to store crystallization screening blocks
 that contain PEG 3350?
 Is it a good idea to freeze the PEG solutions at -80°C and thaw them
 before use?
 Would the freeze-thaw process considerably alter the PEG chain lengths?

 Thank you,
 -Jerome

 Jerome Nwachukwu




 --
 [This e-mail message may contain privileged, confidential and/or
 proprietary information of H3 Biomedicine. If you believe that it has been
 sent to you in error, please contact the sender immediately and delete the
 message including any attachments, without copying, using, or distributing
 any of the information contained therein. This e-mail message should not be

 interpreted to include a digital or electronic signature that can be used
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 reflect an intention to be bound to any legally-binding agreement or
 contract.]

Re: [ccp4bb] Proper detwinning?

[Chris Fage]

Hi Everyone,

Thank you for your helpful input.. Below is a summary of my findings so far.

In P1, P21, or P212121, translational pseudo-symmetry is not detected,
as the max off-origin peak is only 3% of the origin.

Pointless still suggests twinning with [L] of ~0.18 and L^2 of
~0.17 after processing and scaling in P21 with different b edges:
105.6   71.7  200.9
71.7  105.6   201.0
71.1  200.9   105.6

For the P1 unit cell, there are faint but present reflections along
(0,k,0) at k = n. There are clearly no absences along (h,0,0), but I
do see strong absences along (0,0,l) at l = n.

I've noticed that my geometry is not so great when performing twin
refinement in Refmac5 with NCS (~90.8% Ramachandran favored, ~1.7%
outliers). Adding a weighting term of 0.3 (no NCS) seems to improve
these (~95.0% favored, ~0.7% outliers) at the cost of Rwork/Rfree
(from 15.5%/18.3% to 18.6%/21.5%). The same treatment with NCS enabled
counteracts the geometry improvements (~91.1% favored, ~1.6% outliers)
and does not really affect Rwork/Rfree.

Thanks,
Chris

On 7/12/14, Isupov, Michail m.isu...@exeter.ac.uk wrote:
 Dear Chris,

 If you have pseudo-translation in your data close to (0,0,0.5), for
 instance,
 you will have origin ambiguity and, therefore, two P212121 space groups
 differing by a choice of origin. When ZANUDA reports P212121 as a correct
 space group
 it may be not the space group you have submitted. This is true only if you
 have pseudo-translation,
 which can be detected by calculation of the native Patterson.

 To check for it, in ccp4i choose
 'GENERATE PATTERSON' from 'EXPERIMENTAL PHASING/HEAVY METAL LOCATION'
 submenu.
 Choose
 Run FFT to generate PATTERSON option and
 look at at the PEAKMAX output at the end of the log file

 If there are peaks higher than 15 % of  the origin (553.48 in the table
 below),
 you have pseudo-translation, In the example below peak at (0.5,0.5,0) is
 around 30 % of the origin.


  111  553.48 0   0   0   0.  0.  0. 0.00
 0.00   0.00
  2   24   14  171.73   132 132   0   0.5000  0.5000  0.   100.96
 100.96   0.00
  344   10.20 6   2   0   0.0231  0.0084  0. 4.66
 1.69   0.00

 Alternatively, many programs, including MOLREP report pseudo-translation.
 Origin ambiguity int the orthorhombic space group may happen when
 pseudo-translation is close to half of one of your cell parameters.


 If you do not have such pseudo-translation, your case is more complex and do
 not waste time on ZANUDA.

 If you have pseudo-translation, try to refine the model output by ZANUDA in
 P212121.

 Best,

 Misha

 
 From: Chris Fage [cdf...@gmail.com]
 Sent: 12 July 2014 00:33
 To: Isupov, Michail
 Cc: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Proper detwinning?

 Nat and Misha,

 Thank you for the suggestions.

 Xtriage does indeed detect twinning in P1, reporting similar values
 for |L|, L^2, and twin fraction as in P212121.

 The unit cell dimensions for the 2.0-A structure (P1) are:
 72.050  105.987  201.142  89.97  89.98  89.94 P 1

 The unit cell dimensions for the 2.8-A structure (P212121) are:
 75.456  115.154  202.022  90.00  90.00  90.00 P 21 21 21

 I have been processing in HKL2000, which only recognizes one set of
 unit cell parameters for each Bravais lattice (does anyone know how to
 change this?). Specifically, for a primitive monoclinic unit cell it
 estimates:
 104.53  71.82  200.99  89.86  91.80  91.16
 This is the unit cell which refined to Rwork/Rfree ~ 27%/34%.

 Indexing in mosflm gives three options for primitive monoclinic:
 105.6  71.7 200.9  90.0  90.1  90.0
 71.7 105.6  201.0  90.0  89.9  90.0
 71.7 200.9  105.6  90.0  90.3  90.0
 Attempting to integrate in any of these space groups leads to a fatal
 error in subroutine MASKIT. I can also use the index multiple
 lattices feature to get a
 whole slew of potential space group; however, integrating reflections
 leads to the same fatal error.

 Finally, Zanuda tells me that P212121 is the best space group,
 according to R-factors. However, I do not believe P212121 is the
 correct assignment.

 Best,
 Chris


 On 7/10/14, Isupov, Michail m.isu...@exeter.ac.uk wrote:
 I would recommend to run ZANUDA in the default mode from ccp4i or on CCP4
 web server.
 ZANUDA has resolved several similar cases for me.

 Misha

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage
 [cdf...@gmail.com]
 Sent: 10 July 2014 01:14
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Proper detwinning?

 Hi Everyone,

 Despite modelling completely into great electron density, Rwork/Rfree
 stalled at ~38%/44% during refinement of my 2.0-angstrom structure
 (P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning,
 with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447.
 However, there are no twin laws in this space group. I reprocessed

Re: [ccp4bb] Proper detwinning?

[Chris Fage]

Nat and Misha,

Thank you for the suggestions.

Xtriage does indeed detect twinning in P1, reporting similar values
for |L|, L^2, and twin fraction as in P212121.

The unit cell dimensions for the 2.0-A structure (P1) are:
72.050  105.987  201.142  89.97  89.98  89.94 P 1

The unit cell dimensions for the 2.8-A structure (P212121) are:
75.456  115.154  202.022  90.00  90.00  90.00 P 21 21 21

I have been processing in HKL2000, which only recognizes one set of
unit cell parameters for each Bravais lattice (does anyone know how to
change this?). Specifically, for a primitive monoclinic unit cell it estimates:
104.53  71.82  200.99  89.86  91.80  91.16
This is the unit cell which refined to Rwork/Rfree ~ 27%/34%.

Indexing in mosflm gives three options for primitive monoclinic:
105.6  71.7 200.9  90.0  90.1  90.0
71.7 105.6  201.0  90.0  89.9  90.0
71.7 200.9  105.6  90.0  90.3  90.0
Attempting to integrate in any of these space groups leads to a fatal
error in subroutine MASKIT. I can also use the index multiple
lattices feature to get a
whole slew of potential space group; however, integrating reflections
leads to the same fatal error.

Finally, Zanuda tells me that P212121 is the best space group,
according to R-factors. However, I do not believe P212121 is the
correct assignment.

Best,
Chris


On 7/10/14, Isupov, Michail m.isu...@exeter.ac.uk wrote:
 I would recommend to run ZANUDA in the default mode from ccp4i or on CCP4
 web server.
 ZANUDA has resolved several similar cases for me.

 Misha

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage
 [cdf...@gmail.com]
 Sent: 10 July 2014 01:14
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Proper detwinning?

 Hi Everyone,

 Despite modelling completely into great electron density, Rwork/Rfree
 stalled at ~38%/44% during refinement of my 2.0-angstrom structure
 (P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning,
 with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447.
 However, there are no twin laws in this space group. I reprocessed the
 dataset in P21 (8 monomers/AU), which did not alter Rwork/Rfree, and
 in P1 (16 monomers/AU), which dropped Rwork/Rfree to ~27%/32%. Xtriage
 reported the pseudo-merohedral twin laws below.

 P21:
 h, -k, -l

 P1:
 h, -k, -l;
 -h, k, -l;
 -h, -k, l

 Performing intensity-based twin refinement in Refmac5 dropped
 Rwork/Rfree to ~27%/34% (P21) and ~18%/22% (P1). Would it be
 appropriate to continue with twin refinement in space group P1? How do
 I know I'm taking the right approach?

 Interestingly, I solved the structure of the same protein in P212121
 at 2.8 angstroms from a different crystal. Rwork/Rfree bottomed out at
 ~21%/26%. One unit cell dimension is 9 angstroms greater in the
 twinned dataset than in the untwinned.

 Thank you for any suggestions!

 Regards,
 Chris

[ccp4bb] Proper detwinning?

[Chris Fage]

Hi Everyone,

Despite modelling completely into great electron density, Rwork/Rfree
stalled at ~38%/44% during refinement of my 2.0-angstrom structure
(P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning,
with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447.
However, there are no twin laws in this space group. I reprocessed the
dataset in P21 (8 monomers/AU), which did not alter Rwork/Rfree, and
in P1 (16 monomers/AU), which dropped Rwork/Rfree to ~27%/32%. Xtriage
reported the pseudo-merohedral twin laws below.

P21:
h, -k, -l

P1:
h, -k, -l;
-h, k, -l;
-h, -k, l

Performing intensity-based twin refinement in Refmac5 dropped
Rwork/Rfree to ~27%/34% (P21) and ~18%/22% (P1). Would it be
appropriate to continue with twin refinement in space group P1? How do
I know I'm taking the right approach?

Interestingly, I solved the structure of the same protein in P212121
at 2.8 angstroms from a different crystal. Rwork/Rfree bottomed out at
~21%/26%. One unit cell dimension is 9 angstroms greater in the
twinned dataset than in the untwinned.

Thank you for any suggestions!

Regards,
Chris

Re: [ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

Hi Everyone,

Thank you for the advice, especially Pavel's. My issue has been
resolved. I lowered the occupancy and B-factors of the metal ions in
the .pdb and ran phenix.refine for 10 cycles. This removed most of the
negative Fo-Fc density. Anisotropic refinement of the metal ion
B-factors was also effective, dropping Rwork and Rfree by ~1% each. To
do this, I edited the Individual B-factor refinement values as
follows.
Isotropic atoms: not (element Zn)
Anisotropic atoms: element Zn

Best,
Chris


On 5/6/14, Chris Fage cdf...@gmail.com wrote:
 I have used CNS before, but not for this sort of refinement. I see in
 the bindividual.inp file that I can select atoms to be included--it
 is defaulted at known and not hydrogen. Do you know the proper
 nomenclature for selecting a Zn ion in chains A and B?

 Thanks,
 Chris

 On 5/6/14, Steven Herron sherron_...@yahoo.com wrote:

 Refining the occupancy will help your R-factor and flatten your density,
 but you need to be careful to also refine the B-factor of the metal
 ion.  Don't refine both the occupancy and the B-factor during the same
 run (the two are correlated at this resolution), refine the occupancy of
 just the metal ion and then refine the B-factor of just the metal ion
 (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
 easy to refine the occupancy (or B-factor) of just the metal ion.  After
 a few rounds of refinement both parameters will stop changing and you
 will have your answer.  The final B-factor of the metal ion should be
 similar to the amino acid residues that are coordinated to it.

 Soaking in several different ion concentrations and collecting
 additional datasets is also a good idea (if you have the time).  I did
 this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr 4,
 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
 several different Ca2+ ion concentrations and was able to determine the
 binding affinity for that calcium ion using crystallography.

 To make sure I was not stuck in a local minima, I would modify either
 the occupancy of the B-factor of the metal while keeping the other fixed
 and do a refinement.  I even tried both large and small changes (both
 increases and decreases in value).  It always came back to the earlier
 answer.

 Different Ca2+ ion concentrations can give some additional insight into
 the metal binding site.  Between the no-Ca2+ structure and the high-Ca2+
 structure there was a conserved Asp-residue that changed conformation.
 So, I soaked in the appropriate amount of Ca2+ to see the residue in
 both positions.  There was a high correlation between the asp residue
 orientation and the Ca2+ ion occupancy.

 Steven Herron
 sherron_...@yahoo.com




 On 5/6/2014 11:02 AM, Chris Fage wrote:
 Hi Everyone,

 In my 2.5-angstrom structure, there is negative Fo-Fc density
 surrounding a metal ion after refining in Phenix. From anomalous
 diffraction I am certain of the metal's identity and position in each
 monomer. Also, the ion is appropriately coordinated by nearby side
 chains. Should I be refining the occupancy of the ion in attempt to
 flatten the negative density? I am considering soaking the metal ion
 into crystals or cocrystallizing and collecting additional datasets.

 Thanks for your help!

 Regards,
 Chris

Re: [ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

Hi Tim,

That's a good question. The negative density vanished and the model
itself has not changed much. I still plan to set up
soaking/cocrystallization experiments with the metal ion. I am hoping
this will increase occupancy. That is the only solution that comes to
mind.

Regards,
Chris

On 5/8/14, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
 Hi Chris,

 this certainly improved your model with respect to the data including
 all their errors. How do you know this did not make your model worse
 with respect to chemistry, respectively to what is inside your crystal?

 Best,
 Tim

 On 05/08/2014 07:26 PM, Chris Fage wrote:
 Hi Everyone,

 Thank you for the advice, especially Pavel's. My issue has been
 resolved. I lowered the occupancy and B-factors of the metal ions in
 the .pdb and ran phenix.refine for 10 cycles. This removed most of the
 negative Fo-Fc density. Anisotropic refinement of the metal ion
 B-factors was also effective, dropping Rwork and Rfree by ~1% each. To
 do this, I edited the Individual B-factor refinement values as
 follows.
 Isotropic atoms: not (element Zn)
 Anisotropic atoms: element Zn

 Best,
 Chris


 On 5/6/14, Chris Fage cdf...@gmail.com wrote:
 I have used CNS before, but not for this sort of refinement. I see in
 the bindividual.inp file that I can select atoms to be included--it
 is defaulted at known and not hydrogen. Do you know the proper
 nomenclature for selecting a Zn ion in chains A and B?

 Thanks,
 Chris

 On 5/6/14, Steven Herron sherron_...@yahoo.com wrote:

 Refining the occupancy will help your R-factor and flatten your
 density,
 but you need to be careful to also refine the B-factor of the metal
 ion.  Don't refine both the occupancy and the B-factor during the same
 run (the two are correlated at this resolution), refine the occupancy
 of
 just the metal ion and then refine the B-factor of just the metal ion
 (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
 easy to refine the occupancy (or B-factor) of just the metal ion.
 After
 a few rounds of refinement both parameters will stop changing and you
 will have your answer.  The final B-factor of the metal ion should be
 similar to the amino acid residues that are coordinated to it.

 Soaking in several different ion concentrations and collecting
 additional datasets is also a good idea (if you have the time).  I did
 this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr
 4,
 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
 several different Ca2+ ion concentrations and was able to determine the
 binding affinity for that calcium ion using crystallography.

 To make sure I was not stuck in a local minima, I would modify either
 the occupancy of the B-factor of the metal while keeping the other
 fixed
 and do a refinement.  I even tried both large and small changes (both
 increases and decreases in value).  It always came back to the earlier
 answer.

 Different Ca2+ ion concentrations can give some additional insight into
 the metal binding site.  Between the no-Ca2+ structure and the
 high-Ca2+
 structure there was a conserved Asp-residue that changed conformation.
 So, I soaked in the appropriate amount of Ca2+ to see the residue in
 both positions.  There was a high correlation between the asp residue
 orientation and the Ca2+ ion occupancy.

 Steven Herron
 sherron_...@yahoo.com




 On 5/6/2014 11:02 AM, Chris Fage wrote:
 Hi Everyone,

 In my 2.5-angstrom structure, there is negative Fo-Fc density
 surrounding a metal ion after refining in Phenix. From anomalous
 diffraction I am certain of the metal's identity and position in each
 monomer. Also, the ion is appropriately coordinated by nearby side
 chains. Should I be refining the occupancy of the ion in attempt to
 flatten the negative density? I am considering soaking the metal ion
 into crystals or cocrystallizing and collecting additional datasets.

 Thanks for your help!

 Regards,
 Chris





 --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A

[ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

Hi Everyone,

In my 2.5-angstrom structure, there is negative Fo-Fc density
surrounding a metal ion after refining in Phenix. From anomalous
diffraction I am certain of the metal's identity and position in each
monomer. Also, the ion is appropriately coordinated by nearby side
chains. Should I be refining the occupancy of the ion in attempt to
flatten the negative density? I am considering soaking the metal ion
into crystals or cocrystallizing and collecting additional datasets.

Thanks for your help!

Regards,
Chris

Re: [ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

I have used CNS before, but not for this sort of refinement. I see in
the bindividual.inp file that I can select atoms to be included--it
is defaulted at known and not hydrogen. Do you know the proper
nomenclature for selecting a Zn ion in chains A and B?

Thanks,
Chris

On 5/6/14, Steven Herron sherron_...@yahoo.com wrote:

 Refining the occupancy will help your R-factor and flatten your density,
 but you need to be careful to also refine the B-factor of the metal
 ion.  Don't refine both the occupancy and the B-factor during the same
 run (the two are correlated at this resolution), refine the occupancy of
 just the metal ion and then refine the B-factor of just the metal ion
 (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
 easy to refine the occupancy (or B-factor) of just the metal ion.  After
 a few rounds of refinement both parameters will stop changing and you
 will have your answer.  The final B-factor of the metal ion should be
 similar to the amino acid residues that are coordinated to it.

 Soaking in several different ion concentrations and collecting
 additional datasets is also a good idea (if you have the time).  I did
 this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr 4,
 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
 several different Ca2+ ion concentrations and was able to determine the
 binding affinity for that calcium ion using crystallography.

 To make sure I was not stuck in a local minima, I would modify either
 the occupancy of the B-factor of the metal while keeping the other fixed
 and do a refinement.  I even tried both large and small changes (both
 increases and decreases in value).  It always came back to the earlier
 answer.

 Different Ca2+ ion concentrations can give some additional insight into
 the metal binding site.  Between the no-Ca2+ structure and the high-Ca2+
 structure there was a conserved Asp-residue that changed conformation.
 So, I soaked in the appropriate amount of Ca2+ to see the residue in
 both positions.  There was a high correlation between the asp residue
 orientation and the Ca2+ ion occupancy.

 Steven Herron
 sherron_...@yahoo.com




 On 5/6/2014 11:02 AM, Chris Fage wrote:
 Hi Everyone,

 In my 2.5-angstrom structure, there is negative Fo-Fc density
 surrounding a metal ion after refining in Phenix. From anomalous
 diffraction I am certain of the metal's identity and position in each
 monomer. Also, the ion is appropriately coordinated by nearby side
 chains. Should I be refining the occupancy of the ion in attempt to
 flatten the negative density? I am considering soaking the metal ion
 into crystals or cocrystallizing and collecting additional datasets.

 Thanks for your help!

 Regards,
 Chris

Re: [ccp4bb] Trouble Refining Ligand in Phenix

[Chris Fage]

Actually, that was the issue. I manually reset the occupancies of the
problematic atoms in the PDB to unity. This solved my problem.

Thanks to Nat and Tom.

Best,
Chris

On 4/21/14, tom.p...@csiro.au tom.p...@csiro.au wrote:
 Hello Chris,

 This probably isn't your problem, but I once put some atoms into difference
 density (in Coot) and for some reason the occupancy was set to zero, so
 although everything was refined, the density was the same as before (massive
 difference density).  You might want to just check.
 cheers, tom

 Tom Peat
 Biophysics Group
 CSIRO, CMSE
 343 Royal Parade
 Parkville, VIC, 3052
 +613 9662 7304
 +614 57 539 419
 tom.p...@csiro.au
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage
 [cdf...@gmail.com]
 Sent: Tuesday, April 22, 2014 8:21 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Trouble Refining Ligand in Phenix

 Hi Everyone,

 I am trying to refine a structure with a phosphorylated amino acid. After
 refining in Phenix, the Fo-Fc density (green) overlaps the 2Fo-Fc density
 for all atoms of the derivatized amino acid in Coot, almost as if I had not
 built in the residue. I am loading a .cif for the derivative when I run
 phenix.refine. I have also tried ReadySet, but when I click the Run in
 phenix.refine button, I see the message

 Error interpreting command line argument as parameter definition:
 refine_65-coot-2.metal.edits
 RuntimeError: Unexpected end of output.

 Am I just seeing noise, or is Phenix not actually refining this portion of
 the model? I would appreciate any suggestions.

 Best,
 Chris

[ccp4bb] Trouble Refining Ligand in Phenix

[Chris Fage]

Hi Everyone,

I am trying to refine a structure with a phosphorylated amino acid. After
refining in Phenix, the Fo-Fc density (green) overlaps the 2Fo-Fc density
for all atoms of the derivatized amino acid in Coot, almost as if I had not
built in the residue. I am loading a .cif for the derivative when I run
phenix.refine. I have also tried ReadySet, but when I click the Run in
phenix.refine button, I see the message

Error interpreting command line argument as parameter definition:
refine_65-coot-2.metal.edits
RuntimeError: Unexpected end of output.

Am I just seeing noise, or is Phenix not actually refining this portion of
the model? I would appreciate any suggestions.

Best,
Chris

[ccp4bb] High Rwork/Rfree vs. Resolution

[Chris Fage]

Dear CCP4BB Users,

I recently collected a number of datasets from plate-shaped crystals
that diffracted to 1.9-2.0 angstroms and yielded very nice electron
density maps. There is no major density unaccounted for by the model;
however, I am unable to decrease Rwork and Rfree beyond ~0.25 and
~0.30, respectively. Probably due to the more 2-dimensional nature of
my crystals, there is a range of phi angles in which the reflections
are smeared, and I am wondering if the problem lies therein.

I would be grateful if anyone could provide advice for improving my
refinement statistics, as I was under the impression that the
R-factors should be ~5% lower for the given resolution.

A few more pieces of information:
-Space group = P21, with 2 monomers per asymmetric unit;
-Chi square = 1.0-1.5;
-Rmerge = 0.10-0.15;
-Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine;
-PHENIX Xtriage does not detect twinning, but hints at possible weak
translational pseudosymmetry;
-I was previously able to grow one atypically thick crystal which
diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22.
Unfortunately, the completeness of the dataset was only ~90%.

Regards,
Chris

Re: [ccp4bb] High Rwork/Rfree vs. Resolution

[Chris Fage]

Thanks for the assistance, everyone.

For those who suggested XDS: I forgot to mention that I have tried Mosfim,
which is also better than spot fitting than HKL2000. How does XDS compare
to Mosflm in this regard?

I am not refining the high R-factor structure with NCS options. Also, my
unit cell dimensions are 41.74 A, 69.27 A, and 83.56 A, so there isn't one
particularly long axis.

I'm guessing the low completeness of the 1.65 angstrom dataset has to do
with obstacles the processing software encountered on a sizable wedge of
frames (there were swaths of in red in HKL2000). I'm not sure why this
dataset in particular was less complete than the others.

Thanks,
Chris


On Fri, Feb 21, 2014 at 6:41 PM, Chris Fage cdf...@gmail.com wrote:

 Dear CCP4BB Users,

 I recently collected a number of datasets from plate-shaped crystals
 that diffracted to 1.9-2.0 angstroms and yielded very nice electron
 density maps. There is no major density unaccounted for by the model;
 however, I am unable to decrease Rwork and Rfree beyond ~0.25 and
 ~0.30, respectively. Probably due to the more 2-dimensional nature of
 my crystals, there is a range of phi angles in which the reflections
 are smeared, and I am wondering if the problem lies therein.

 I would be grateful if anyone could provide advice for improving my
 refinement statistics, as I was under the impression that the
 R-factors should be ~5% lower for the given resolution.

 A few more pieces of information:
 -Space group = P21, with 2 monomers per asymmetric unit;
 -Chi square = 1.0-1.5;
 -Rmerge = 0.10-0.15;
 -Data were processed in HKL2000 and refined in Refmac5 and/or
 phenix.refine;
 -PHENIX Xtriage does not detect twinning, but hints at possible weak
 translational pseudosymmetry;
 -I was previously able to grow one atypically thick crystal which
 diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22.
 Unfortunately, the completeness of the dataset was only ~90%.

 Regards,
 Chris

Re: [ccp4bb] Phasing with Many Monomers/AU

[Chris Fage]

I am grateful for all of the suggestions. I think I have enough tricks to
try at this point, but I may check back with this group if things don't
work out.

Many thanks once again,
Chris


On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote:

 Hello Everyone,

 I am currently trying to phase a structure with an asymmetric unit
 predicted to contain 20-24 monomers (space group P1). The native crystals,
 while beautiful in appearance (see attached), only diffract to ~3.4-3.0
 angstroms at best, and SeMet-derived crystals grow with poor morphology
 (small needles). Also, based a fluorescence scan, I know that mercury does
 not bind appreciably. Other than screening for a new space group, what
 options might I have for phasing this many monomers at lower resolution? Is
 there any real chance of solving the structure in this space group?

 Thank you in advance for any suggestions!

 Regards,
 Chris

Re: [ccp4bb] Phasing with Many Monomers/AU

[Chris Fage]

Thank you all for your responses. I already have a few ideas about how to
approach the problem.

One of my concerns with so monomers per asymmetric unit at lower resolution
was the failure of MR software. Neither PHENIX nor Phaser MR have made
progress. I am fairly new to anomalous methods, having solved only two
structures by SeMet-based SAD. I've certainly picked up on a number of
tricks from the recent messages on heavy atoms, but I thought my case might
be a little unusual. I am confident the space group is P1, as it was the
only viable option when I indexed four clean albeit low-res datasets.

The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a
synchrotron.

The conditions for both native and SeMet crystals are:
8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5.

Macromolecular seeding of native crystals into SeMet drops yields the
needle-like crystals.

Any further input is greatly appreciated!

Regards,
Chris


On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote:

 Hello Everyone,

 I am currently trying to phase a structure with an asymmetric unit
 predicted to contain 20-24 monomers (space group P1). The native crystals,
 while beautiful in appearance (see attached), only diffract to ~3.4-3.0
 angstroms at best, and SeMet-derived crystals grow with poor morphology
 (small needles). Also, based a fluorescence scan, I know that mercury does
 not bind appreciably. Other than screening for a new space group, what
 options might I have for phasing this many monomers at lower resolution? Is
 there any real chance of solving the structure in this space group?

 Thank you in advance for any suggestions!

 Regards,
 Chris

[ccp4bb] Phasing with Many Monomers/AU

[Chris Fage]

Hello Everyone,

I am currently trying to phase a structure with an asymmetric unit
predicted to contain 20-24 monomers (space group P1). The native crystals,
while beautiful in appearance (see attached), only diffract to ~3.4-3.0
angstroms at best, and SeMet-derived crystals grow with poor morphology
(small needles). Also, based a fluorescence scan, I know that mercury does
not bind appreciably. Other than screening for a new space group, what
options might I have for phasing this many monomers at lower resolution? Is
there any real chance of solving the structure in this space group?

Thank you in advance for any suggestions!

Regards,
Chris
attachment: Crystals.jpg