[ccp4bb] Position in membrane protein drug discovery

[Sandra Jacob]

We have an opening for a protein scientist to work in drug discovery projects 
where integral membrane proteins are the targets. Please see more information 
about the position and how to apply under the following link: 
https://www.novartis.com/careers/career-search/job-details/271919BR
Best regards,
Sandra.



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Re: [ccp4bb] Extra density close to phosphate bound to Zn2+

[Bernhard Rupp]

One practically HAS to do what Dale describes. The sigma levels of a difference 
map peak depends on how much it is above the mean noise level of the map. With 
high resolution (and given a cooperating molecule) it is very likely that the 
model is very good and complete, thus low mean map noise level, and anything 
missing will create huge peaks. A forgotten water molecule then can easily have 
difference peak (sigma not absolute) levels in the 20s... 

Best, BR

-Original Message-
From: CCP4 bulletin board  On Behalf Of Dale Tronrud
Sent: Tuesday, August 6, 2019 00:24
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Extra density close to phosphate bound to Zn2+

   One thing to remember is that Li+ only has two electrons.  It should be a 
little more than twice the density of an hydrogen (because the charge pulls in 
the electron cloud).  If you are seeing lithium at 8 "sigma" you should be 
seeing the well located hydrogen atoms at 4 "sigma".  Are you?

   I always like controls.  Take out one of the near by water molecules and see 
how that, known, difference peak compares to your mystery peak.
 A Li+ should have about 1/5th the height of an H2O.  If you like the 
hypothesis of two orientations of the PO4 group, the relative height of the two 
peaks will give insight to the occupancy ratio.

   Remember, if the PO4 has two orientations in the crystal, the water 
molecules it (they?) is/are bound to will likely also have alternatives.

Dale Tronrud

On 8/5/2019 6:05 AM, Maria Håkansson wrote:
> Dear CCP4 bulletin board,
> 
> I am working with some lytic enzymes called endolysines, which bind 
> Zn2+ in the active site. I have three homologues protein determined to 
> 1.2 Å each where the Zn2+ is bound to a cystein, two histidines and 
> one phosphate ion added (1.9-2.3 Å binding distances) in the 
> crystallization experiment.
> 
> Now to my question. Close to the phosphate (B-factor=20Å2) ion a 8 
> sigma peak is present in all three endolysines, see below.
> I have modeled it to a Na+ (B-factor= 30 Å2) or a Li+ (B factor = 
> 13Å2) ion.
> Sodium has benn added in the crystallization experiments since sodium 
> potassium phosphate salt has been used. The only reason for including 
> Li+ is that I think the binding distances (1.7-2.0 Å) are too short 
> for Na+.
> 
> I have also tried to make a model with the phosphate in two different 
> conformations but it does not fit.
> 
> Have anyone seen something similar before? What is the most correct 
> way of dealing with unknown densities?
> It is difficult to disregard +8 sigma difference density close to the 
> active site.
> 
> Thanks in advance for any help!
> 
> Best regards,
> Maria
> 
>  
> 
> Maria Håkansson, PhD, Crystallization Facility Manager Principal 
> Scientist
> 
> SARomics Biostructures AB
> Medicon Village
> SE-223 81 Lund, Sweden
> 
> Mobile: +46 (0)76 8585706
> Web: www.saromics.com 
> 
> 
> 
> 
> 
> --
> --
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
> 



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[ccp4bb] Waters and controls

[Alwyn Jones]

Dale Tronrud recently said '  I always like controls.’ in the discussion 
concerning water molecules.
So do I but I prefer an alternative to his suggestion, which is the profile of 
how the average carbonyl oxygen would look in an F11 map. This is absolute in 
the sense that it is full occupancy and it is something truly integral to the 
molecule of interest. My original implementation built a 3D profile based on 
the local density of 50 carbonyl oxygens that had been chopped from the phases 
(the water commands in ‘A-Z of O’ from my home page if you are interested in 
details). There is a much simpler variation on this theme that works with any 
refinement program and any graphics program where you set zero occupancy to 
three consecutive carbonyl oxygens ( I use residues in a b-strand) at the 
beginning of the refinement and keep them like that until you are finished (is 
one ever finished?). If you go through multiple rebuilding rounds, it pays to 
regularise the segment every cycle or two, since the C can move towards the 
correct position of the O, otherwise it is easy to do. Just remember to reset 
the occupancies before submitting coordinates to the PDB and then carry out a 
final refinement run.

The peak height is, of course, resolution dependent but any graphics program 
should allow you to change a slider to see what is realistic.
Although this gives an absolute value for what you are looking for, the 
crystallographer still has to decide what fraction of the average carbonyl 
oxygen shall I use for adding waters :)

Seeing three gleaming beacons in your F11 map focuses your mind and may dampen 
unrealistic expectations.

Sorry for the long post...

Alwyn Jones
al...@xray.bmc.uu.se
http://xray.bmc.uu.se/alwyn



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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Clemens Vonrhein]

Dear Ivan,

On Mon, Aug 05, 2019 at 05:44:56PM -0400, Ivan Shabalin wrote:
> Dear Kay,
> 
> Thanks a lot for your answers!
> 
> To my best understanding, REFMAC does not have an option of for restoring
> reflections only in certain resolution shells.

Remember that you can always do some simple (but powerfull) operations
within SFTOOLS (*). So using something like

  read refmac.mtz
  CALC col resol = 0.5 stol /
  select col FP = absent
  absent col FWT  if col resol < 3.0
  absent col PHWT if col resol < 3.0
  delete col resol
  select all
  write refmac_new.mtz
  
might/should do the trick: setting all filled-in FWT/PHWT values to a
MNF (missing-number-flag) for reflections with resolution higher than
3A. So you should only have filled-in 2mFo-DFc valeus for the low-res
data (3A and lower).

But check those commands above (typed from memory)

Cheers

Clemens

(*) there is very little one can't do with SFTOOLS



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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Eleanor Dodson]

There are certainly some problems with REFMAC after an anisotropy
correction. The FreeR flags and D corrections are organised in spherical
shells, whereas the D aniso volume is elliptical..


As Kay points out the REFMAC map coefficient for a difference map is 0 if
there is no measurement so there is no bias in those maps.

the 2mFo-DFc maps do set the map coefficient to D Fc if there is no
measurement, potentially introducing bias.

The thinking is that when a large low resolution term is missing it is
better to approximate it to something realistic, rather than set it to 0.
For high weak resolution data D Fc will also probably be insignificant and
there would be very little gain by including it, and the possibility of
introducingbias..

However nowadays the data collection techniques usually provide pretty
complete data at low resolution,

On Tue, 6 Aug 2019 at 07:44, Kay Diederichs 
wrote:

> Dear Ivan,
>
> one thing I forgot to mention: I think the difference maps should not show
> model bias due to "fill-in". This is because the term D*Fcalc that is
> filled in as a replacement for m*Fobs is just compensated by the term D*Fc
> that is subtracted when forming the m*Fobs - D*Fcalc difference
> coefficients.
> This means that when building the model, you can let yourself be guided
> primarily by the difference maps. These will not suffer from model bias due
> to fill-in.
>
> I like the idea of the "shaping the MTZ file" that Robbie suggests, but I
> still need to work out the proper sftools commands, like those at
> http://staraniso.globalphasing.org/test_set_flags_about.html .
>
> best,
> Kay
>
> On Mon, 5 Aug 2019 17:44:56 -0400, Ivan Shabalin <
> iva...@iwonka.med.virginia.edu> wrote:
>
> >Dear Kay,
> >
> >Thanks a lot for your answers!
> >
> >To my best understanding, REFMAC does not have an option of for
> >restoring reflections only in certain resolution shells. But, it should
> >not be a problem for datasets with good completeness in low resolution
> >shells. Also, refinement against intensities is available only for twin
> >refinement.
> >
> >I will take this as a conclusion for datasets with good low resolution
> >completeness: "even if the visual effect of weak reflections on the map
> >may be low, the errors in the model coordinates will be less if the weak
> >amplitudes are used in refinement"
> >
> >Thanks!
> >
> >Ivan
> >
> >
> >With best regards,
> >Ivan Shabalin, Ph.D.
> >Research Scientist,
> >Department of Molecular Physiology and Biological Physics,
> >University of Virginia,
> >1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
> >Charlottesville, VA 22908
> >https://www.linkedin.com/in/shabalinig/
> >https://minorlab.org/person/ivan_s/
> >
> >On 8/3/19 04:32, Kay Diederichs wrote:
> >> Dear Ivan,
> >>
> >> On Thu, 1 Aug 2019 22:10:36 -0400, Ivan Shabalin <
> iva...@iwonka.med.virginia.edu> wrote:
> >>
> >>> Dear CCP4BB,
> >>>
> >>> There seems to be a general consensus for extending data to higher
> >>> resolution to include as much meaningful data as possible. "Meaningful"
> >>> can be defined in different ways. I heard/read opinions such as 0.5
> >>> CC1/2, 0.3 CC1/2, 0.15 CC1/2,
> >>
> >> This is numerology - why not 0.3 or 0.12345?  The EM community has
> agreed on the "gold standard" of 0.143 for FSC which has a similar
> definition as CC1/2 - this value is chosen because the quantity analogous
> to CC* is then 0.5 !
> >>
> >>> and stepped (paired) refinement. The
> >>> latter seems to be one of the most rigorous options according to many
> >>> crystallographers.
> >>>
> >>> Including more data sounds like a good thing, but, it sometimes results
> >>> in low completeness in high resolution shells. As far as i understand,
> >>> this may result from:
> >>>
> >>> a) anisotropic diffraction (if a software cuts of resolution in
> >>> non-isotropic way)
> >>>
> >>> b) sub-optimal data collection (e.g. due to limitations of the
> >>> instrument, such as minimum detector distance allowed, absence of
> kappa,
> >>> limits on oscillation range)
> >>>
> >>> In the commonly referred paper, the completeness is 96% in the highest
> >>> shell (Karplus, P. A., & Diederichs, K. (2012). Linking
> crystallographic
> >>> model and data quality. Science (New York, N.Y.), 336(6084),
> 1030–1033.)
> >>> In other words, these tests were performed for an almost complete
> dataset.
> >>>
> >>> I used to think that more data is always better, but, as I learned
> >>> recently from Clemens Vonrhein, the resulting low completeness may
> cause
> >>> model bias in the maps.
> >>
> >> ... due to "fill-in" of missing reflections which is performed by
> Refmac and phenix.refine (
> https://www.phenix-online.org/documentation/faqs/refine.html#general);
> don't know about other programs.
> >>
> >>> Indeed, REFMAC by default tries to restore missing reflections, which
> >>> are approximated as DFc
> >>> (
> https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html
> ).
> >>>
> >>>
> >>> W

Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Ivan Shabalin]

Dear Clemens,

Thanks!

It sounds like a good test to do on the output .mtz and see if there are 
any significant changes in maps after these "filled in" coefficients are 
removed.


Ivan

With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/

On 8/6/19 08:59, Clemens Vonrhein wrote:

Dear Ivan,

On Mon, Aug 05, 2019 at 05:44:56PM -0400, Ivan Shabalin wrote:

Dear Kay,

Thanks a lot for your answers!

To my best understanding, REFMAC does not have an option of for restoring
reflections only in certain resolution shells.


Remember that you can always do some simple (but powerfull) operations
within SFTOOLS (*). So using something like

   read refmac.mtz
   CALC col resol = 0.5 stol /
   select col FP = absent
   absent col FWT  if col resol < 3.0
   absent col PHWT if col resol < 3.0
   delete col resol
   select all
   write refmac_new.mtz
   
might/should do the trick: setting all filled-in FWT/PHWT values to a

MNF (missing-number-flag) for reflections with resolution higher than
3A. So you should only have filled-in 2mFo-DFc valeus for the low-res
data (3A and lower).

But check those commands above (typed from memory)

Cheers

Clemens

(*) there is very little one can't do with SFTOOLS







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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Pavel Afonine]

For the record, phenix.refine always produces two versions of 2mFo-DFc
maps, with and without filling in missing Fobs. The one that opens in Coot
by default is the "filled" map, but you can always load the other one for
comparisons.

Pavel

On Tue, Aug 6, 2019 at 8:34 AM Ivan Shabalin 
wrote:

> Dear Clemens,
>
> Thanks!
>
> It sounds like a good test to do on the output .mtz and see if there are
> any significant changes in maps after these "filled in" coefficients are
> removed.
>
> Ivan
>
> With best regards,
> Ivan Shabalin, Ph.D.
> Research Scientist,
> Department of Molecular Physiology and Biological Physics,
> University of Virginia,
> 1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
> Charlottesville, VA 22908
> https://www.linkedin.com/in/shabalinig/
> https://minorlab.org/person/ivan_s/
>
> On 8/6/19 08:59, Clemens Vonrhein wrote:
> > Dear Ivan,
> >
> > On Mon, Aug 05, 2019 at 05:44:56PM -0400, Ivan Shabalin wrote:
> >> Dear Kay,
> >>
> >> Thanks a lot for your answers!
> >>
> >> To my best understanding, REFMAC does not have an option of for
> restoring
> >> reflections only in certain resolution shells.
> >
> > Remember that you can always do some simple (but powerfull) operations
> > within SFTOOLS (*). So using something like
> >
> >read refmac.mtz
> >CALC col resol = 0.5 stol /
> >select col FP = absent
> >absent col FWT  if col resol < 3.0
> >absent col PHWT if col resol < 3.0
> >delete col resol
> >select all
> >write refmac_new.mtz
> >
> > might/should do the trick: setting all filled-in FWT/PHWT values to a
> > MNF (missing-number-flag) for reflections with resolution higher than
> > 3A. So you should only have filled-in 2mFo-DFc valeus for the low-res
> > data (3A and lower).
> >
> > But check those commands above (typed from memory)
> >
> > Cheers
> >
> > Clemens
> >
> > (*) there is very little one can't do with SFTOOLS
> >
>
> 
>
> 
>
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>



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[ccp4bb] 3DNA-DSSR annotated G-quadruplexes in the PDB (http://G4.x3dna.org)

[Xiang-Jun Lu]

The website http://g4.x3dna.org for the DSSR annotated G-quadruplexes (G4)
in the PDB has been launched. Here are two examples showcasing G4 features
automatically derived by DSSR: http://g4.x3dna.org/entries/6r9l and
http://g4.x3dna.org/entries/5ua3

The 3DNA-DSSR program detects and annotates G4 structures from atomic
coordinates in PDB or PDBx/mmCIF format. It identifies G-tetrads and
arranges them into G4 helices, composed of G4 stems via coaxial stacking
interactions. G4 stems are categorized by loops connecting the four
strands, and by common names [e.g., chair, basket] or the Webba da Silva
nomenclature [e.g., 3(-P-Lw-Ln) for 2GKU]. DSSR accounts for bugles along a
strand, characterizes G4 structures using rigid-body parameters (including
Rise and Twist), and quantifies stacking by overlapping areas. The program
also introduces a novel schematic representation, highlighting G-tetrads
using square blocks (to render in PyMOL).

I've have been actively developing and refining the DSSR module for G4
structures for more than two years. I greatly appreciate your comments or
suggestions.

Best regards,

Xiang-Jun

[This announcement is cross-posted on PDB-l and CCP4BB]

--
Xiang-Jun Lu (Ph.D.)
Email: xiang...@x3dna.org
Web: http://x3dna.org/
Forum: http://forum.x3dna.org/



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[ccp4bb] FFT bug, CCP4i job file configuration

[Roger Rowlett]

FYI,

When FFT is run in the CCP4i interface, the output file destination is
configured incorrectly. Examining the batch file that is created for the
job, it appears that CCP4i ignores the MAPOUT filename and substitutes the
file name of the temporary file, which is (alas) deleted in the last step.
There is a workaround by using the full file path in the MAPOUT line
instead of the project directory and local file name.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu



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Re: [ccp4bb] How to include in refinement high resolution shells with VERY low completeness ?

[Ivan Shabalin]

Pavel,

Thanks for pointing that out.

Based on one dataset, it seems to me that using this kind of maps 
"no-fill" as an additional guide can help tracing some side-chains.


Ivan

With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/

On 8/6/19 12:17, Pavel Afonine wrote:
For the record, phenix.refine always produces two versions of 2mFo-DFc 
maps, with and without filling in missing Fobs. The one that opens in 
Coot by default is the "filled" map, but you can always load the other 
one for comparisons.


Pavel

On Tue, Aug 6, 2019 at 8:34 AM Ivan Shabalin 
mailto:iva...@iwonka.med.virginia.edu>> 
wrote:


Dear Clemens,

Thanks!

It sounds like a good test to do on the output .mtz and see if there
are
any significant changes in maps after these "filled in" coefficients
are
removed.

Ivan

With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
https://www.linkedin.com/in/shabalinig/
https://minorlab.org/person/ivan_s/

On 8/6/19 08:59, Clemens Vonrhein wrote:
 > Dear Ivan,
 >
 > On Mon, Aug 05, 2019 at 05:44:56PM -0400, Ivan Shabalin wrote:
 >> Dear Kay,
 >>
 >> Thanks a lot for your answers!
 >>
 >> To my best understanding, REFMAC does not have an option of for
restoring
 >> reflections only in certain resolution shells.
 >
 > Remember that you can always do some simple (but powerfull)
operations
 > within SFTOOLS (*). So using something like
 >
 >    read refmac.mtz
 >    CALC col resol = 0.5 stol /
 >    select col FP = absent
 >    absent col FWT  if col resol < 3.0
 >    absent col PHWT if col resol < 3.0
 >    delete col resol
 >    select all
 >    write refmac_new.mtz
 >
 > might/should do the trick: setting all filled-in FWT/PHWT values to a
 > MNF (missing-number-flag) for reflections with resolution higher than
 > 3A. So you should only have filled-in 2mFo-DFc valeus for the low-res
 > data (3A and lower).
 >
 > But check those commands above (typed from memory)
 >
 > Cheers
 >
 > Clemens
 >
 > (*) there is very little one can't do with SFTOOLS
 >





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[ccp4bb] IST Austria cryo-EM inauguration symposium, October 18

[Carrie Bernecky]

Dear colleagues,

We cordially invite you to attend the IST Austria cryo-EM inauguration 
symposium, which will take place on October 18, 2019. Please join us for 
some exciting talks and posters. Registration is free and we have a 
lineup of great speakers: Jan Pieter Abrahams,
Peter Brzezinski, Gaia Pigino, Jürgen Plitzko, Henning Stahlberg, Sharon 
Wolf, and Peijun Zhang.


More details about the symposium can be found at: https://cryo-em.ist.ac.at/

With two new transmission electron microscopes (Thermo Scientific 
Glacios and Titan Krios), a Thermo Scientific Aquilos cryo-focused ion 
beam/scanning electron microscope, and a dedicated cryo-correlative 
Leica CLEM setup, IST has established Austria’s largest cryo-EM center. 
The aim of this symposium is to celebrate the opening of our expanded 
facility and to highlight some of the exciting research involving 
cryo-electron microscopy.


We plan to host a poster session in the afternoon, and encourage 
everyone interested to participate. Abstract submission will be done 
through the registration page. We will also offer a tour of the EM 
facility in the afternoon. We hope many of you can join us!


Best wishes on behalf of the organizing team,
Carrie Bernecky
Florian Schur
Leonid Sazanov

--
Carrie Bernecky
Institute of Science and Technology Austria
Am Campus 1
3400 Klosterneuburg
Austria



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