Re: [ccp4bb] what happened to Molprobity

[Mark J. van Raaij]

same here, the website http://molprobity.biochem.duke.edu/index.php 
 loads, but when trying uploading 
a pdb file nothing happens.
Hopefully it is just a temporary glitch.

Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 30 Apr 2024, at 13:25, Gerlind Sulzenbacher 
>  wrote:
> 
> Dear all,
> 
> The Molprobity server appears to be not functional from my side. I just 
> installed the latest version of Phenix and the module Molprobity has 
> dissapeared.
> 
> Can anybody provide information on the latest evolutions of this so precious 
> tool?
> 
> Thanking you in advance, with best wishes,
> 
> Gerlind
> 
> 
> 
> 
> -- 
> Gerlind Sulzenbacher
> Architecture et Fonction des Macromolécules Biologiques
> UMR7257 CNRS, Aix-Marseille Université
> Case 932
> 163 Avenue de Luminy
> 13288 Marseille cedex 9
> France
> Tel +33 413 94 95 27
> E-mail: gerlind.sulzenbac...@univ-amu.fr
> 
> 
> 
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Re: [ccp4bb] The experiment is still very much needed (though AlphaFold helps a lot)

[Mark J. van Raaij]

just came across this critique of that paper on Twitter:

https://twitter.com/Robert_Palgrave/status/1730358675523424344 
Robert Palgrave (@Robert_Palgrave) on X
twitter.com

https://twitter.com/Robert_Palgrave/status/1730358675523424344but I'm not 
enough of an expert to judge - perhaps some characterizations were wrong and a 
lot of the paper does stand.


> On 1 Dec 2023, at 20:51, Bryan Lepore  wrote:
> 
> Adding to that literature list a bit outside :
> 
> Merchant, A., Batzner, S., Schoenholz, S.S. et al. 
> 
> Quote:
> 
> "... we show that graph networks trained at scale can reach unprecedented 
> levels of generalization, improving the efficiency of materials discovery by 
> an order of magnitude. "
> 
> Scaling deep learning for materials discovery. 
> 
> Nature (2023), November
> 
> https://doi.org/10.1038/s41586-023-06735-9
> 
> 
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Re: [ccp4bb] how to increase b factor for water in protein- ligand crystal structure

[Mark J. van Raaij]

Dear Mandar,

- you may be able to find some more, less well-ordered, waters by looking at 
(difference) maps. These will probably refine to have higher B-factors, pushing 
your average up.
- some of your best-ordered water molecules may in fact be metal ions, you can 
check the coordination. Modeling these as metal will then remove some waters 
with likely very low B-factors, also leading to a higher average B-factor for 
the remaining water molecules. Check My Metal may be useful: 
https://cmm.minorlab.org

But:
- it shouldn't really be a goal to increase the B-factor of the solvent, but to 
find and model all the water molecules (metal ions, protein and everything 
else) with reasonable density and hydrogen bonds.
- when you write "improve the data", I assume you mean "improve the model".

Best wishes,

Mark

Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 2 Nov 2023, at 08:24, Mandar Bhutkar 
>  wrote:
> 
> Hi everyone,
> I am working on one of the x-ray diffraction data. Refinement details are 
> given below. My solvent b factor is 18.44. Can anyone pls suggest me how to 
> improve the data? 
> Thank you. 
> -- 
> Mr. Mandar Bhutkar,
> Ph.D. student,
> Molecular Virology lab,
> IIT Roorkee.
> Resolution range
> 23.06–2.60
> Space group
> P 2 21 21
> Unit cell dimensions: a b c (Å)
> α, β, γ (°)
> 51.6  60.7 184.36
> 90  90   90
> Completeness (%)
> 99.3
> Rmerge a
> 0.372
> I/σ(I)
> 2.67
> CC(1/2)
> 0.773
> Refinement
> Reflections used in refinement
> 10011
> Reflections used for R-free
> 924
> R-workb
> 0.231
> R-freeb
> 0.273
> Wilson B-factor (Å)
> 28.9
> Number of non-hydrogen atoms
> 4302
> Macromolecules
> 4086
> Ligands
> 94
> Solvent
> 124
> Protein residues
> 508
> RMS (bonds) (Å)c
> 0.01
> RMS (angles) (˚) c
> 1.89
> Ramachandran Plot
>  
> Favored (%)
> 97.83
> Allowed (%)
> 1.38
> Outliers (%)
> 0.79
> Average B-factor (Å)
> 28.0
> macromolecules (Å)
> 28.65
> ligands (Å)
> 48.98
> solvent (Å)
> 18.44
> 
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Re: [ccp4bb] Database for submitting unprocessed diffraction data

[Mark J. van Raaij]

This months publication in Acta Cryst F by Loes Kroon-Batenburg may be of 
interest:
https://journals.iucr.org/f/issues/2023/10/00/va5053/index.html 



Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 18 Oct 2023, at 18:23, JEROME JOHNSON 
>  wrote:
> 
> Hello all,
> 
> As the subject says, I am looking for an open online database to store 
> unprocessed diffraction data.  Does such a database exist? Essentially we 
> would like our diffracted data to be freely available online while also 
> outsourcing some of our data management.
> 
> Any advice would be wonderful!
> 
> Best,
> Jerome
> 
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Re: [ccp4bb] Assistant Professor in Structural Biology, University of Nebraska-Lincoln

[Mark J. van Raaij]

Dear Gerlind,

Even though I am also not completely familiar with the US system, I think the 
position is for more than one year. Indefinite if you get tenure after a 
certain time (perhaps after five years). In this case the first five years are 
secure.
But the Univ only pays you for 9 months out of 12 each year. You can then pay 
yourself out of a grant for the other 3 months - if you get a grant that allows 
paying yourself of course.
Still seems a bit unfair, but at least it is not just a 9 months one time 
contract.

Best wishes,

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 15 Oct 2023, at 17:15, Gerlind Sulzenbacher 
>  wrote:
> 
> Dear Mark,
> 
> thank you for posting this job offer, probably very interesting for people 
> currently unemployed. 
> 
> Personally, I feel that working conditions and job offers in the scientific 
> field have deteriorated considerably in recent years.
> I've always been shocked by one-year job offers involving international 
> movements.
> Now we seem to be down at job offers for 9 months.
> From a human point of view, I find this extremely worrying.
> 
> Sorry to pollute the mailing list with my personal opinions.
> All the best.
> 
> Gerlind
> 
> 
> On 15/10/2023 16:46, Mark Wilson wrote:
>> Dear Colleagues, 
>>  
>> The Department of Biochemistry at the University of Nebraska-Lincoln (UNL) 
>> invites applications for a tenure-track nine-month (academic year) faculty 
>> position at the rank of Assistant Professor. Areas of particular interest 
>> include but are not limited to time-resolved approaches to understanding 
>> macromolecular function, multiscale imaging, and integrated computational 
>> and experimental approaches to structural biology. Researchers at UNL have 
>> collaborations with national user facilities pioneering time-resolved X-ray 
>> diffraction techniques and have established a state-of-the-art cryo-EM 
>> facility housing a Thermo Scientific Glacios 200 keV Cryo TEM equipped with 
>> a Falcon 4i direct electron detector camera, a Selectris zero-loss energy 
>> filter, and capability for micro-electron diffraction of crystalline 
>> samples. The new cryo-EM facility is part of a strategic growth plan at UNL 
>> and complements existing strengths in X-ray crystallography, biophysics, and 
>> computation at UNL.
>>  
>> Review of applications will begin November 15, 2023 and continue until the 
>> position is filled or the search is closed. To view details of the position 
>> and the application, go to https://employment.unl.edu 
>> <https://employment.unl.edu/>, requisition F_230175 or visit 
>> https://employment.unl.edu/postings/88408. Click “Apply for this Job” and 
>> complete the information form. As an EO/AA employer, the University of 
>> Nebraska considers qualified applicants for employment without regard to 
>> race, color, ethnicity, national origin, sex, pregnancy, sexual orientation, 
>> gender identity, religion, disability, age, genetic information, veteran 
>> status, marital status, and/or political affiliation. See 
>> https://www.unl.edu/equity/notice-nondiscrimination.
>>  
>>  
>> Best regards,
>> Mark
>>  
>> Mark A. Wilson (he/him)
>> Professor
>> Department of Biochemistry/Redox Biology Center
>> University of Nebraska
>> N118 Beadle Center
>> 1901 Vine St.
>> Lincoln, NE 68588
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
> 
> -- 
> Gerlind Sulzenbacher
> Architecture et Fonction des Macromolécules Biologiques
> UMR7257 CNRS, Aix-Marseille Université
> Case 932
> 163 Avenue de Luminy
> 13288 Marseille cedex 9
> France
> Tel +33 413 94 95 27
> E-mail: gerlind.sulzenbac...@univ-amu.fr 
> <mailto:gerlind.sulzenbac...@univ-amu.fr>
> 
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Re: [ccp4bb] the structures of Nucleic acid

[Mark J. van Raaij]

This just appeared and may be relevant:
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad726/7272628

Critical Reviews and Perspectives
When will RNA get its AlphaFold moment? 

Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 18 Sep 2023, at 18:07, William G. Scott 
> <2844d921eb97-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> The phosphorus absorption edge is about 5.8Å.
> 
> I've had much better luck with 5-Br-U for anomalous phasing.
> 
> Molecular replacement with sub-structural fragments can also work:
> 
> 
> 
> Yours sincerely,
> 
> William G. Scott
> Professor, Department of Chemistry and Biochemistry
> and The Center for the Molecular Biology of RNA
> University of California at Santa Cruz
> Santa Cruz, California 95064  
> USA
> 
>> On Sep 18, 2023, at 2:43 AM, Eleanor Dodson 
>> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
>> 
>> I am afraid most scientists will use the most straightforward technique! 
>> If SAD is available the PHOSPHATE backbone of DNA will provide sufficient 
>> signal to allow SAD to work, and you get an unambiguous answer to whether it 
>> is A-DNA or B or Z...
>> MR will usually work of course as well
>> Eleanor
>> 
>> 
>> On Mon, 18 Sept 2023 at 09:18, Natesh Ramanathan  
>> wrote:
>> Dear Fu Xingke,
>> 
>> Depends on what Nucleic Acid you are talking of.  If it is RNA, you 
>> can expect some sequence to tertiary structure correspondence so you might 
>> be able to try more MR as compared to DNA.   DNA may have double helical 
>> architecture but less sequence to tertiary structure correspondence, and 
>> hence DNA is less likely to have a 3D structure like RNA specific structure 
>> for a sequence.
>> 
>> SAD has become a straight forward method to avoid all these problems 
>> to get ab-initio structure.  So many go for it directly.
>> 
>> Hope that helps.
>> Best wishes,
>> Natesh
>> 
>> On Mon, 18 Sept 2023 at 13:36, fuxingke  wrote:
>> Dear Colleagues,
>> 
>> Reacently, I find the structures of Nucleic acid are solved by 
>> single-wavelength anomalous diffraction(SAD). So, why molecular replacement 
>> (MR) not?
>> 
>> Regards
>> 
>> 
>> 
>> Best wishes,
>> 
>> Fu Xingke
>> 
>> Institute of Physics CAS
>> 
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>> 
>> 
>> 
>> -- 
>> --
>> "Live Simply and do Serious Things .. "
>> - Dorothy Mary Crowfoot Hodgkin OM, FRS
>> 
>> "In Science truth always wins"
>> - Max Ferdinand Perutz OM FRS
>> --
>> Dr. Ramanathan Natesh
>> Associate Professor, 
>> School of Biology and Center for High-Performance Computing (CHPC),
>> Founding and Current President of Cryo Electron Microscopy and 3 Dimensional 
>> Image Processing Society of India (CEM3DIPSI),
>> Indian Institute of Science Education and Research Thiruvananthapuram 
>> (IISER-TVM),
>> Maruthamala P.O., Vithura,
>> Thiruvananthapuram,  695551, Kerala, India
>> 
>> nat...@iisertvm.ac.in
>> http://faculty.iisertvm.ac.in/natesh
>> 
>> Researcher ID: http://www.researcherid.com/rid/C-4488-2008
>> ORCID: http://orcid.org/-0002-1145-5962
>> Vidwan-ID : 94134: http://iisertvm.irins.org/profile/94134
>> PUBLONS: https://publons.com/author/1520837/ramanathan-natesh#profile
>> 
>> Office Ph. 0091- 471-2778087
>> 
>> 
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> 
> 
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Re: [ccp4bb] Crystallisation textbook

[Mark J. van Raaij]

Dear All,

The Acta Cryst F special issue is also a very good resource (although I admit 
that I am biased).
Can be read online here:
https://journals.iucr.org/special_issues/2016/crystallization/ 
<https://journals.iucr.org/special_issues/2016/crystallization/>
or ordered as a book from the IUCr here:
https://www.iucr.org/publications/iucr/buy

Best wishes,

Mark J van Raaij (Section Editor of Acta Cryst F)
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 14 Jul 2023, at 16:19, Hough, Michael (DLSLtd,RAL,LSCI) 
> <69715b1ac6c0-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi all,
>  
> I'd be grateful for any recommendations of a good up-to-date textbook 
> covering macromolecular crystallisation. Currently we are using the excellent 
> book by Terese Bergfors (2009) but would be interested if there is anything 
> more recent with discussion around automation, micro-crystallisation and so on
>  
> Many thanks in advance for your help!
>  
> Kind regards,
>  
> Mike 
>  
> -- 
> 
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[ccp4bb] Two postdoctoral positions open at CNB-CSIC

[Mark J. van Raaij]

Two postdoctoral positions open at CNB-CSIC

Two fully funded postdoctoral positions in the area of Structural Biology are 
available at Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.

PIs: Carmen San Martín, Mark J. van Raaij
Starting on: June 2023
Contract length: 3 years
Subject: The intelligent design of more efficient adenovirus vectors using 
structural data.

Tasks: To solve structures of adenovirus particles or adenovirus proteins, 
alone or in complex with hosts factors. To analyze structures and design 
changes in proteins to tailor more favorable virus host interactions for 
efficient targeting and gene delivery.

Requested skills:

Position A: demonstrated experience in protein structure determination. 
Additional experience in virus structure, protein crystallography or 
cryo-electron microscopy strongly desired.

Position B: demonstrated experience in protein design / protein-protein 
interactions. Additional experience in virus structure or protein structure 
determination strongly desired.

Requirements for both positions:

Applicants should hold a PhD degree in biochemistry, structural biology, 
biophysics, protein chemistry or a related discipline.

Ability to work independently and efficiently as part of a team.
Well organized with attention to detail and excellent record keeping. Strong 
communication skills and fluency in written and spoken English. Enthusiasm, 
motivation, and capability to learn new methods. Problem-solving skills.
Ability to work in an international collaborative research setting.

How to apply: please send to i...@cnb.csic.es a PDF document containing: a 
cover letter describing your previous research achievements and motivation to 
apply; your detailed CV including a list of publications and explaining your 
own contribution; contact information for at least two references (ideally 
including your PhD supervisor). Please indicate whether you are applying to 
Position A or B. 




Mark van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)




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Re: [ccp4bb] Renumber residues working PDB file - Applying a sequences numbering to PDB file

[Mark J. van Raaij]

I guess you could also get pdbset to do it in one go, using various lines 
(being careful to use the right order and not “overwrite” residues with the 
same name).
Ideally you’d want to run only the renumber part of a auto-build program like 
buccaneer or arp-warp, without the rebuilding or refinement, might be a nice 
little addition to ccp4.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 5 Feb 2023, at 08:03, Eleanor Dodson 
> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Usually you can bully coot into doing it but by bit. Say you need to renumber 
> A 1-8. I often have to change the chain id to Z say then renumber Z. And so 
> on . Then go back once you have finished and reset chain id fir Z1-8 to A. 
> Tedious but possible! 
> 
> Or just run a few cycles of buccaneer with your structure - buccaneer should 
> do the job fir you...
> Eleanor
> 
> On Sat, 4 Feb 2023 at 22:11, Matt McLeod  <mailto:mjmcleo...@gmail.com>> wrote:
> Hi all,
> 
> I have been refining a structure and somehow along the way the residue 
> numbers have completely shifted.  For instance, the first section of residues 
> are shifted by say 8 numbers, then there is a gap from where the resnumbers 
> go from 121, 151, 152, 153...and so on.  Its quite the mess.
> 
> Is there a way to take a sequence file with residue numbers correct and apply 
> these to the PDB file?  I have tried Renumber Residues in coot but this isnt 
> working, it just shifts some of them and does opposite shifts elsewhere since 
> its so discontinuous. Align and mutate just shifts them incorrectly from the 
> inputted sequence without applying the sequence file number to the PDB.
> 
> Any suggestions would be appreciated before I go and do this all manually...
> Matt
> 
> 
> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[Mark J. van Raaij]

PS it’s probably not a salt crystal…TCEP is not a salt, your ligand I presume 
is also not a salt, a small cleaved peptide neither. As to why previously in a 
very similar condition you did get your desired protein plus (other) ligand 
crystal, it just means the molecule (TCEP') crystallises in a similar condition 
to your protein - I don’t think you can conclude much more than that (unless 
there is some other difference like the TCEP being older this time and more 
oxidised, for example).

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 4 Feb 2023, at 15:48, kavyashreem  wrote:
> 
> Dear all, 
> 
> Sorry for the confusion created, I did not mean that a protein would have fit 
> in the small unit cell. My question was -
> 
> 1. Why are there closely spaced spots arising in salt crystal?
> 
> 2. If TCEP could crystallize in the condition, I have got a protein (same as 
> this)+ligand (different ligand) complex in very close condition. (ligand size 
> is within 500Da).
> 
> Thank you
> 
> Kavya
> 
> 
> 
> On 2023-02-03 14:35, a.perra...@nki.nl wrote:
> 
>> Hi Kavya,
>>  
>> Try https://csb.wfu.edu/tools/vmcalc/vm.html 
>> <https://csb.wfu.edu/tools/vmcalc/vm.html> 
>>  
>> This tells you that a 30kD protein simply does not fit the cell.
>>  
>> I am pretty sure you crystallised the ligand, or TCEP actually.
>>  
>> Also, if you look at the diffractions pattern, its clear the crystal 
>> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
>> 2.0A.
>>  
>>  
>> 
>>> On 3 Feb 2023, at 09:22, kavyashreem >> <mailto:kavyashr...@instem.res.in>> wrote:
>>> Dear all,
>>> 
>>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>>> condition 10%PEG3350, 50mM Zinc acetate.
>>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 
>>> 8. 
>>> Crystal: Crystal:   
>>> crystal under UV m
>>> <8ef9453e.png>
>>> When we collected the data at an in-house facility, it looked something 
>>> like this:
>>> 
>>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>>> I have not come across a protein diffraction like this, nor of a salt. When 
>>> I ran the gel for the incubated protein (protein+ligand), there was no 
>>> degradation.
>>> Although, I was sure there is some problem with this image I tried 
>>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not 
>>> the third.
>>> Can anyone please shed some light on this diffraction image?
>>> How can it happen?
>>>  
>>> Thank you
>>> Regards
>>> Kavya
>>>  
>>> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[Mark J. van Raaij]

the unit cell in the c direction is quite long, 49 Å, this gives the relatively 
close spots in one direction.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 4 Feb 2023, at 15:48, kavyashreem  wrote:
> 
> Dear all, 
> 
> Sorry for the confusion created, I did not mean that a protein would have fit 
> in the small unit cell. My question was -
> 
> 1. Why are there closely spaced spots arising in salt crystal?
> 
> 2. If TCEP could crystallize in the condition, I have got a protein (same as 
> this)+ligand (different ligand) complex in very close condition. (ligand size 
> is within 500Da).
> 
> Thank you
> 
> Kavya
> 
> 
> 
> On 2023-02-03 14:35, a.perra...@nki.nl wrote:
> 
>> Hi Kavya,
>>  
>> Try https://csb.wfu.edu/tools/vmcalc/vm.html 
>> <https://csb.wfu.edu/tools/vmcalc/vm.html> 
>>  
>> This tells you that a 30kD protein simply does not fit the cell.
>>  
>> I am pretty sure you crystallised the ligand, or TCEP actually.
>>  
>> Also, if you look at the diffractions pattern, its clear the crystal 
>> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
>> 2.0A.
>>  
>>  
>> 
>>> On 3 Feb 2023, at 09:22, kavyashreem >> <mailto:kavyashr...@instem.res.in>> wrote:
>>> Dear all,
>>> 
>>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>>> condition 10%PEG3350, 50mM Zinc acetate.
>>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 
>>> 8. 
>>> Crystal: Crystal:   
>>> crystal under UV m
>>> <8ef9453e.png>
>>> When we collected the data at an in-house facility, it looked something 
>>> like this:
>>> 
>>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>>> I have not come across a protein diffraction like this, nor of a salt. When 
>>> I ran the gel for the incubated protein (protein+ligand), there was no 
>>> degradation.
>>> Although, I was sure there is some problem with this image I tried 
>>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not 
>>> the third.
>>> Can anyone please shed some light on this diffraction image?
>>> How can it happen?
>>>  
>>> Thank you
>>> Regards
>>> Kavya
>>>  
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
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Re: [ccp4bb] Regarding the diffraction image

[Mark J. van Raaij]

like others mentioned, looks like something in between a salt and a protein, 
perhaps TCEP, the ligand, a peptide cleaved from your protein by trace protease.
If possible, I would move the detector closer, collect an atomic resolution 
dataset and try to solve the structure by direct methods. You never know, it 
could be something interesting.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/
https://namedrop.io/markvanraaij

> On 3 Feb 2023, at 09:22, kavyashreem  wrote:
> 
> Dear all,
> 
> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
> condition 10%PEG3350, 50mM Zinc acetate.
> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8. 
> Crystal: Crystal:   
> crystal under UV m
> <8ef9453e.png>
> When we collected the data at an in-house facility, it looked something like 
> this:
> 
> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
> I have not come across a protein diffraction like this, nor of a salt. When I 
> ran the gel for the incubated protein (protein+ligand), there was no 
> degradation.
> Although, I was sure there is some problem with this image I tried 
> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
> third.
> Can anyone please shed some light on this diffraction image?
> How can it happen?
>  
> Thank you
> Regards
> Kavya
>  
> 
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Re: [ccp4bb] Low resolution and high anisotropy

[Mark J. van Raaij]

The improvement in statistics by STARANISO is really spectacular, but I do 
wonder how much is caused by the directionally variable resolution limits and 
how much by the removal of solvent rings? The original data set statistics have 
big dips in CC1/2 at 6, 4.3 and 3.7 Å.
Also in the final statistics table, the completeness is really high, but I 
guess reflections from the "bad" direction(s) are not taken into account? It 
would be good to also know the spherical completeness and the CC1/2 values 
before going too far ahead with these data. Or perhaps better phrased: I'd 
simultaneously try to get a dataset from a better flash-cooled crystal while 
trying to get the most out of this dataset.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 24 Oct 2022, at 03:43, Xu, Shenyuan  wrote:
> 
> Dear CCP4 community,
> 
> I have encountered a dataset, which I thought should be easy to solve. The 
> volume of the cell unit seems to be expanded after image 271, which I think 
> is caused by radiation damage. After removing the last few images, the scaled 
> statistics seem good with the resolution set at 3:07 A:
> 
> d_max  d_min   #obs  #uniq   mult.  %comp r_mrg   r_meas 
>r_pim   r_anom   cc1/2   cc_ano
>  91.54   8.33   3086   11972.58  98.44 345.251.30.101
> 0.1330.0860.145   0.973*  -0.148
>   8.33   6.61   3005   12252.45  99.51 256.527.30.160
> 0.2120.1380.260   0.923*  -0.140
>   6.61   5.78   2883   11902.42  98.43  92.9 9.40.278
> 0.3680.2370.461   0.541*  -0.165
>   5.78   5.25   3195   12172.63  99.10  75.6 8.10.283
> 0.3690.2330.436   0.618*  -0.107
>   5.25   4.87   3192   12062.65  99.42  92.5 8.30.281
> 0.3670.2320.427   0.856*  -0.187
>   4.87   4.59   3230   12132.66  99.02 118.410.30.288
> 0.3780.2400.456   0.870*  -0.053
>   4.59   4.36   2797   11562.42  92.93 137.012.20.309
> 0.4100.2660.496   0.843*  -0.239
>   4.36   4.17   2694   11182.41  93.17 280.216.20.419
> 0.5750.3920.847   0.491*  -0.142
>   4.17   4.01   3102   11882.61  95.50 133.6 9.90.392
> 0.5160.3300.622   0.663*  -0.268
>   4.01   3.87   3224   12052.68  98.69 120.4 7.30.406
> 0.5390.3490.687   0.791*  -0.126
>   3.87   3.75   2939   11812.49  98.91  90.7 6.80.491
> 0.6520.4250.810   0.691*  -0.042
>   3.75   3.64   1981   10211.94  82.34  87.3 5.00.557
> 0.7560.5060.885   0.540*  -0.018
>   3.64   3.54   2374   10822.19  89.13 194.611.20.502
> 0.6750.4470.973   0.625*  -0.237
>   3.54   3.46   2622   11222.34  92.35 310.510.10.432
> 0.5850.3920.851   0.603*  -0.154
>   3.46   3.38   17399451.84  76.58  48.2 3.60.930
> 1.2470.8221.542   0.444*  -0.046
>   3.38   3.31   2847   12402.30  98.57  85.4 4.20.691
> 0.9300.6161.242   0.523*  -0.060
>   3.31   3.24   2838   11532.46  97.88  70.8 3.30.693
> 0.9340.6201.341   0.362*   0.006
>   3.24   3.18   3097   12122.56  97.66  71.4 2.50.692
> 0.9240.6051.173   0.526*   0.044
>   3.18   3.12   3204   12162.63  99.10  79.6 3.70.668
> 0.8860.5761.338   0.440*  -0.081
>   3.12   3.07   3059   11722.61  98.16  73.5 2.40.714
> 0.9450.6121.633   0.362*  -0.177
>  91.50   3.07  57108  232592.46  95.22 138.410.80.383
> 0.5130.3360.671   0.656*  -0.149
> 
> I used Mrbump to do the MR, most sequence identities of the starting 
> templates are more than 0.85, and some of them are structures predicted from 
> alpha fold 2. But after refinement (including jelly-body, proSmart, TLC), the 
> best R/Free R stuck at around 0.42/0.45. Inspecting the electron density map 
> shows that the model does not fit the electron density well. The space group 
> is P1, Cell is 58.49 63.97 91.60 91.71 91.86 99.47, should be 5 or 6 
> molecules in the asymmetric unit.
> 
> I checked the data quality, it said the data is highly anisotropy. Then
> I searched the CCP4 forum and used the STARANISO Server and UCLA server, but 
> still cannot improve the refinement. The data statistics after drawing 
> ellipsoidal resolution limits is good:
> 
>  
>  resolution   observed redundancycomplet

[ccp4bb] EDR (Sophos) and CCP4 compatibility

[Mark J. van Raaij]

Dear CCP4-ers,

After a hacking scare, in our institution all computers connected to the 
internet will have to have EDR software installed for safety against hackers. 
We will get Sophos here, at least on OSX, to be installed directly by IT 
services, not by ourselves, from an admin account on all our computers 
controlled by them.
Is this compatible with CCP4 installation or should I be resigned to having an 
off-line computer for CCP4?
The other point is that IT services will update the operating systems of those 
computers from now on, and I worry about major OSX or Linux updates breaking 
CCP4 and other necessary programs.
Any comments and experience with EDR software in general and Sophos in 
particular is appreciated, both for OSX and Linux.

Saludos cordiales,

Mark


Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/



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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Mark J. van Raaij]

Hi Eike,
we bought a Biorad back in 2005 or so, an Akta Purifier in 2011 and recently an 
Akta Go (2020). All work(ed) well, the latter two are in use. Don't know about 
the Biorad, it's in Santiago de Compostela. Both the Biorad and Purifier have 
been used a lot, the Akta Go not that much so far. Just because the programs 
are set up for the Purifier I think, not because people like it more per se.
For "normal" protein purifications all are fine. We've had breakdowns in both 
the Biorad and Purifier, which could be fixed with not too much spending.
I'm not sure about this kind of equipment in a multi-user environment, best to 
keep it in one group I think, otherwise it's difficult people feel responsible 
enough to use them properly. This is even more important for columns, they are 
easily ruined if just once an incorrectly prepared sample is run through.
Price: we got very good deals on the two Akta systems we bought, not more 
expensive than a comparable Biorad. But Cytiva service/repair is very expensive.
Good luck,
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/
https://namedrop.io/markvanraaij

> On 16 Sep 2022, at 09:39, Schulz, Eike-Christian  
> wrote:
> 
> Dear all, 
>  
> I am considering to purchase a chromatography system for routine protein 
> purification. The device is supposed to be used in a multi-user environment, 
> hence ease of use, ease of training and ease of maintenance is important. I 
> am rather looking for a robust system people like to use than a system that 
> comes with many bells and whistles that no one dares to touch.
>  
> Is there any particularly _bad_ experience with either system?
>  
> Is there any specific advantage of one system over the other?
>  
> Does anyone have experience about (long-term) stability / performance of the 
> systems?
>  
> Do you think the higher price-point of the Aekta systems is justified?
>  
>  
> With best regards, 
>  
> Eike
>  
>  
>  
> 
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Re: [ccp4bb] Unidentified electron density

[Mark J. van Raaij]

Could it be Tris?



Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/
https://namedrop.io/markvanraaij

> On 30 Jun 2022, at 09:03, Sayan Saha  wrote:
> 
> Dear All,
> 
> I am trying to refine a protein structure. I observed an additional electron 
> density (Figures attached) connected to the aspartate residue.
> 
> Any suggestion in this regard would be appreciated.
> 
> With best regards,
> Sayan Saha.
> 
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Re: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide crystallization?

[Mark J. van Raaij]

Hi David,

do you think that alphafold2 dis/order prediction is better than specific 
disorder predictors?

i.e.
http://dis.embl.de <http://dis.embl.de/> , which we've used for construct design
https://prdos.hgc.jp <https://prdos.hgc.jp/> , which I haven't really tried yet
https://iupred2a.elte.hu <http://iupred2a.elte.hu/> (which a colleague here 
likes because it also tries to predict likelihood of protein interactions)

Or perhaps it's just because you were going to run alphafold2 anyway?

best wishes,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)


> On 4 Apr 2022, at 21:30, David Briggs  wrote:
> 
> Hi Scott,
> 
> I've used AF2 order/disorder prediction (based up pLDDT score) to decided 
> upon construct boundaries. We turned a non-expressing construct into a 
> reasonably well expressing construct based on the AF2 prediction. 
> 
> It's part of my construct design process now.
> 
> HTH,
> 
> Dave
> 
> --
> Dr David C. Briggs
> Senior Laboratory Research Scientist
> Signalling and Structural Biology Lab
> The Francis Crick Institute
> London, UK
> ==
> about.me/david_briggs
> From: CCP4 bulletin board  on behalf of Scott Classen 
> 
> Sent: Monday, April 4, 2022 8:06:38 PM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] Has anyone successfully used RoseTTAFold or AF2 to guide 
> crystallization?
>  
>  
> External Sender: Use caution.
>  
> Hello CCP4,
> 
> Has anyone successfully used the available ML/AI protein folding tools to 
> guide crystallization construct design? Maybe you had a protein or domain 
> that was resistant to crystallization efforts and the folding algorithms  
> predicted some loops or termini that were disordered? Then you trimmed or 
> modified them in some way to aid in crystallization? Or if you haven’t done 
> this yourself, are you aware of anyone who has?
> 
> Thanks,
> Scott 
> 
> 
> ~~
> Scott Classen, Ph.D.
> ALS-ENABLE
> TomAlberTron Beamline 8.3.1
> SIBYLS Beamline 12.3.1
> Advanced Light Source
> Lawrence Berkeley National Laboratory
> 1 Cyclotron Rd
> MS6R2100
> Berkeley, CA 94720
> mobile 510.206.4418
> desk 510.495.2697
> beamline 510.495.2134
> ~~
> 
> 
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Re: [ccp4bb] MR solution not working

[Mark J. van Raaij]

if the spacegroup is the same as wt and the cell params are similar, just do 
rigid body refinement and forget about MR (I’ll never understand people running 
MR needlessly in these cases…apart from wasting computer time it risks placing 
the new solution in a different place than the wt)
if the spacegroup is different, check cell parameters in the pdb and/or run 
Contaminer to see if you’ve crystallised a contaminant
try MR with packing restraints relaxed or switched off, just in case the mutant 
has loop in a new orientation that might clash with crystal neighbours in the 
old orientation.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 3 Mar 2022, at 05:43, Shubhashish Chakraborty 
> <750c9a1ca48b-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hello,
> I am trying to solve a dataset using molecular replacement. However, neither 
> Phaser MR nor Molrep can give any solution. 
> In Phaser, I have received an advisory that Top FTF has not packed. 
> I have tried molecular replacement using the wild-type protein at different 
> resolutions (I am working on a mutant).
> Also, I have truncated the loops from the input structure. However, none have 
> worked. 
> So, what can be the possible way to solve this data set?
>  
> Thank you
>  
> Shubhashish Chakraborty
> PhD JRF 2018
> Structural and Molecular Biology Lab (Varma Lab)
> Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
> Khargar, Navi Mumbai
> E-mail: schakrabo...@actrec.gov.in <mailto:schakrabo...@actrec.gov.in>
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Re: [ccp4bb] Negative density

[Mark J. van Raaij]

or perhaps something in the non-visible part of the ligand pulling electrons 
out of the rings? can this somehow be specified as ligand input?
on the other hand, I guess this is near the "end" of refinement, so the Fo-Fc 
difference map is probably very flat - meaning that 3 sigma corresponds to very 
few electrons per cubic Å and may be called "noise".

> On 22 Feb 2022, at 13:01, Jon Cooper 
> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> I thought it was tryptophan but the nitrogen and extra bond are in the wrong 
> places so it must be a ligand and, if so, have you tried refining the 
> occupancy? Cheers, Jon.C.
> 
> 
> Sent from ProtonMail mobile
> 
> 
> 
>  Original Message 
> On 22 Feb 2022, 05:34, S < shine.star1...@gmail.com> wrote:
> 
> Hi All,
> 
> I am getting this negative density in the centre of the ring. Could you 
> please help me with this?
> 
> 
> Resolution: 1.7A
> 2FoFc - 1.5
> FoFc - 3.0
> 
> Thanks in advance.
> Regards,
> Renu
> 
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Re: [ccp4bb] keyword for refmac to output coordinates in cif format

[Mark J. van Raaij]

sorry, you can ignore my question.
The mmcif file is output all along, for some reason I had never noticed it.
how stupid of me...

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 30 Oct 2021, at 01:03, Mark J. van Raaij  wrote:
> 
> Dear All,
> 
> this may be something simple but I can’t find it in the CCP4i GUI or online.
> Is there a keyword to make refmac output the coordinates as a cif file 
> instead of a pdb file - or better, as both?
> Or is it some other program that converts the formats?
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> 
> 
> 
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[ccp4bb] keyword for refmac to output coordinates in cif format

[Mark J. van Raaij]

Dear All,

this may be something simple but I can’t find it in the CCP4i GUI or online.
Is there a keyword to make refmac output the coordinates as a cif file instead 
of a pdb file - or better, as both?
Or is it some other program that converts the formats?

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain



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Re: [ccp4bb] Add hydrogens

[Mark J. van Raaij]

Dear Sam,

1CDW is from 1996, when it was not obligatory (or common practice) to upload 
structure factors to the PDB.
So I think you can't do any refinement, just perhaps some optimisation.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/

> On 29 Sep 2021, at 13:03, Sam Tang  wrote:
> 
> Dear community
> 
> This may appear to be a silly question -- I am trying to add hydrogens to the 
> structure in PDB 1CDW. My initial thought is to run a single run of 
> refinement with a refinement program. It happens that I cannot locate the map 
> coefficients under the entry (am I missing something?) So... is there an easy 
> way to do what I want in this case?
> 
> Warm regards
> 
> Sam
> 
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Re: [ccp4bb] Topology diagrams software

[Mark J. van Raaij]

some info here:
https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Topology_plots
(haven’t done it myself for a while, so not sure which programs are still being 
maintained)

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 10 Jun 2021, at 13:14, Gambelli, Lavinia  wrote:
> 
> Hello, 
> 
> I'm looking for a software to make topology diagrams of proteins. Ideally, 
> I'd like to submit a pdb file and obtain the topology model. Do you know what 
> alternatives are out there? So far I'm only aware of PDBsum that can do this, 
> but I was wondering if there is something else available. 
> 
> Kind regards,
> Lavinia
> 
> Dr. Lavinia Gambelli
> Postdoctoral Researcher
> Office number: 01392 727468
>  
> <http://emps.exeter.ac.uk/physics-astronomy/staff/lg496>http://emps.exeter.ac.uk/physics-astronomy/staff/lg496
>  <http://emps.exeter.ac.uk/physics-astronomy/staff/lg496>
> - - - - - - - - - - - - - - - - - - 
> University of Exeter 
> Living Systems Institute 
> Stocker Road, Exeter, Devon, EX4 4QD, UK
> 
> 
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Re: [ccp4bb] Analysis of NMR ensembles

[Mark J. van Raaij]

Dear Dale,
Aren’t NMR spectroscopists, in contrast to us crystallographers, not in the 
lucky situation though that they should have noticed the absence of terminal 
residues during the assignment phase though? I.e. they would usually have peaks 
for the protons of those residues in the 1D, TOCSY, COSY spectra, even though 
NOEs may be absent.
I agree with you that other reasons than flexibility could cause absence of 
NOE’s, although I think that for well-determined NMR ensembles in almost all 
cases it is indeed flexibility / multiple conformations. If not enough 
restraints have been input, you might get artificial “flexible” regions, and 
obtaining more NOEs, secondary structure restraints, measuring orientational 
restraints should shore these up.
(Assuming that in the previous assignent phase all protons peaks could be 
properly assigned of course).
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 26 May 2021, at 23:06, Dale Tronrud  wrote:
> 
> Dear Boaz,
> 
>   We are likely in agreement. "Deficient NOE's for some regions (e.g. loops) 
> arise from their flexibility, ..."  This makes it sound like you agree that 
> these deficiencies in other regions may be caused by properties other than 
> flexibility.
> 
>   As an extreme example, the N-terminal region of a protein may have a broad 
> distribution in the ensemble model either because this region experiences 
> many conformations in solution, or because this peptide was cleaved from the 
> protein at some earlier time and its absence was not recognized by the 
> experimentalist.
> 
> Dale Tronrud
> 
> On 5/26/2021 1:06 PM, Boaz Shaanan wrote:
>> Hi Dale and Cecil,
>> This is quite a circular argument, isn't it? Deficient NOE's for some 
>> regions (e.g. loops) arise from their flexibility, hence they are not as 
>> well resolved as other (e.g. internal ) regions for which the number of NOE 
>> is large. So they are flexible by all accounts and, not surprisingly, align 
>> usually with high B-factor regions in the corresponding crystal structures. 
>> In cases where such flexible regions are held by crystal contacts the 
>> situations would likely be different.
>> Cheers,
>>Boaz
>> /Boaz Shaanan, Ph.D.
>> Dept. of Life Sciences
>> Ben-Gurion University of the Negev
>> Beer-Sheva 84105
>> Israel
>> E-mail: bshaa...@bgu.ac.il
>> Phone: 972-8-647-2220
>> Fax:   972-8-647-2992 or 972-8-646-1710 /
>> //
>> //
>> /
>> /
>> 
>> *From:* CCP4 bulletin board  on behalf of Dale 
>> Tronrud 
>> *Sent:* Wednesday, May 26, 2021 10:46 PM
>> *To:* CCP4BB@JISCMAIL.AC.UK 
>> *Subject:* Re: [ccp4bb] Analysis of NMR ensembles
>> I agree with Dr Breyton. The variability in an NMR ensemble does not
>> reflect "mobility" but simply "uncertainty" in conformation.  The spread
>> in coordinates in some regions simply reflects the lack of experimental
>> data which could define a single conformation.  There are many reasons
>> why these data are be absent and high mobility is only one.
>> Dale Tronrud
>> On 5/26/2021 8:45 AM, Cécile Breyton wrote:
>>> Hello,
>>> In my understanding of NMR, the loops and terminii that adopt very 
>>> different conformations in the structure ensemble rather reflect the fact 
>>> that for those residues, the number of constraints is lower, thus the 
>>> number of structures that fulfil the constraints is larger A dynamics 
>>> study of the protein will be much more informative.
>>> Cécile
>>> Le 26/05/2021 à 17:29, S. Mohanty a écrit :
>>>> Hi Harry,
>>>> 
>>>> The superpose/overlay of all the structures in PyMol should inform you the 
>>>> rigid part of the protein as well as the flexible part. The rigid part 
>>>> would have very low backbone RMSD or overlay tightly and the flexible part 
>>>> (loops, N-term and C-term etc.) would not superpose tightly. If you check 
>>>> literature, the dynamics of the protein may have been studied through NMR 
>>>> relaxation.
>>>> 
>>>> Smita
>>>> 
>>>> 
>>>> On Wednesday, May 26, 2021, 10:05:05 AM CDT, Harry Powell - CCP4BB 
>>>> <193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:
>>>> 
>>>> 
>>>> Hi
>>>> 
>>>> Given that there are plenty of people on this

Re: [ccp4bb] Unmodeled density

[Mark J. van Raaij]

oh, 1.45 Å is a very short distance for metal coordination.
perchlorate has a Cl to O distance of that length
https://en.wikipedia.org/wiki/Perchlorate

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 26 May 2021, at 15:20, leo john  wrote:
> 
> Hi All:
> 
> Thank You very much for the response and yes it is at symmetry axis. Distance 
> between the bigger blob and smaller ones is approx 1.45 Ang.
> I have tried fitting BO3, BO4, but no luck?
> 
> Thanks 
> John
> 
> On Wed, May 26, 2021 at 2:14 PM Pearce, N.M. (Nick)  <mailto:n.m.pea...@uu.nl>> wrote:
> Is it on a symmetry axis? If so it could be the superposition of two 
> molecules (a molecule and a copy of itself). 
> 
>> On 26 May 2021, at 15:08, leo john > <mailto:ljohn16012...@gmail.com>> wrote:
>> 
>> Hi Group
>> Can you please suggest what this unmodeled blob can be (see appended 
>> picture)?
>> I have Malonate, Boric Acid and Peg in my condition, and crystals were 
>> soaked in GOL.
>> 
>> I have tried fitting PO4 and SO4 so far.
>> 
>> Thank You
>> John
>> 
>> 
>> 
>> 
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Re: [ccp4bb] Unmodeled density

[Mark J. van Raaij]

PS I’d model a metal and 4 waters and then measure the distances after 
refinement. And then look at M Harding's ActaD papers and try to work out which 
coordination configuration and distances work best. And, when you get the 
chance, run an emission spectrum at the beamline to help identify the putative 
heavy(ish) atom that was co-crystallised.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 26 May 2021, at 15:08, leo john  wrote:
> 
> Hi Group
> Can you please suggest what this unmodeled blob can be (see appended picture)?
> I have Malonate, Boric Acid and Peg in my condition, and crystals were soaked 
> in GOL.
> 
> I have tried fitting PO4 and SO4 so far.
> 
> Thank You
> John
> 
> 
> 
> 
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Re: [ccp4bb] Unmodeled density

[Mark J. van Raaij]

the central blob looks too big for B or even for P or S.
Perhaps Zn, Cd? (although you didn’t add it, it might have been in the protein 
buffer or dragged through the purification by the protein?)

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 26 May 2021, at 15:08, leo john  wrote:
> 
> Hi Group
> Can you please suggest what this unmodeled blob can be (see appended picture)?
> I have Malonate, Boric Acid and Peg in my condition, and crystals were soaked 
> in GOL.
> 
> I have tried fitting PO4 and SO4 so far.
> 
> Thank You
> John
> 
> 
> 
> 
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Re: [ccp4bb] sugestions on weak diffracting protein crystals

[Mark J. van Raaij]

If the foldamer(s) have exposed flexible parts they may impede ordered 
crystallisation. Perhaps some minor tweaks to the foldamer or foldamers you are 
co-crystallising with might lead to a better crystal contacts?
For example removing one or a few residues from the end(s) or shortening a loop.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 18 May 2021, at 12:08, Deepak Deepak  wrote:
> 
> Dear all,
> 
> I have got multiple crystals (see picture 1) of a protein (8kDa) with a 
> helical aromatic oligoamide foldamer (5kDa) but these crystals diffract very 
> poorly (see the diffraction pattern in picture 2). 
> 
> I prepare a 1.3mM:1.3mM complex of protein: foldamer in 20mM Tris, pH 7.5 
> buffer. Crystals grew in 3-5 days in sitting and hanging drop at 20 Deg C and 
> 25 Deg C in the following conditions:
> 
> - 20% PEG 400, 0.1M MES pH 6.0
> -20% PEG 400, 0.1M Sodium Cacodylate pH 6.0
> 
> Multiple cryo used were:
> -25%Glycerol in mother solution
>  -30% glycerol in water  
> -30%PEG 400,
> -35% PEG 400
> -20% PEG 8000 + 40% PEG 400 mix
> 
> Kindly suggest some methods/modifications on how can I improve the resolution 
> and get better-diffracting crystals. Please let me know if you need more 
> information.
> 
> Kind regards,
> Deepak
> Ph.D. Student
> 
> PS: The protein is a DNA binding protein and I have crystallized and solved 
> the structure of this protein with its DNA partner and now I crystallized it 
> with our foldamers but diffraction is not good. There are multiple structures 
> of the Protein+DNA complex in literature but no apo-protein structure as the 
> protein needs a binding partner to crystallize. We already have solution 
> studies showing a good binding.
> 
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Re: [ccp4bb] unknown density

[Mark J. van Raaij]

The ring looks too big to be imidazole or a nucleotide or a carbohydrate, so 
it’s probably mainly water molecules.
Perhaps partially replaced by PEG to explain the density between them (i.e. 
water molecules in most copies of the protein and PEG in some other copies). 
I’ve seen horse-shoe shaped PEG in a high-res structure before, PEGs in several 
confirmations might explain a circle.
Practically speaking, I’d first model five waters and see if they refine well.

Mark 

> On 22 Mar 2021, at 14:58, Sam Tang  wrote:
> 
> Hello fellow colleagues
> 
> Hope you are all well while the pandemics persists. I just wonder if anyone 
> may have an idea what this density (looking like a pentagon) might be. The 
> data was collected to 1.8 A and crystal was grown in Bis-tris + PEG3350. 
> Imidazole residual? Nucleotide (the protein itself is nucleotide-binding, but 
> shouldn't be at this particular site)?
> 
> https://drive.google.com/file/d/1L9UBFmW72P214itM2HJR_DVy3FaA6FEZ/view?usp=sharing
>  
> 
> 
> Thanks!
> 
> BRS
> 
> Sam
> 
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Re: [ccp4bb] Can twinning be seen in the diffraction pattern?

[Mark J van Raaij]

Hi Marina,
The close-together spots in the zoom inset of your figure I think are not split 
spots, but separate reflections. They are close togeter because you appear to 
have a unit cell with one axis much longer than the other two (we work on 
elongated proteins, so we have some experience with that). The same short 
distances are also clearly visible in the picture on the bottom left.
If you index the image in MOSFLM and it puts boxes around both I'd conclude 
they are separate reflections, but there are perhaps more sophisticated ways to 
verify this.
So I think its true that in this case the twinning is not (obviously) visible 
in the diffraction pattern - but detected through intensity statistics later.
Best wishes,
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain



> On 12 Mar 2021, at 11:30, Marina Gárdonyi 
>  wrote:
> 
> 
> Hello everyone,
> 
> I am a PhD student at the Philipps-University in Marburg and I am currently 
> writing my thesis.
> 
> I have problems to understand whether in my case twinning can be seen in the 
> diffraction pattern or not.
> 
> I know that it depends on the type of twinning wheter you can see it in the 
> diffraction pattern. The crystal had a resolution of 2.2 A. During processing 
> it seemed to have the space group P622, but in the end it was P3(2)21. With 
> phenix Xtriage I found out, that the data set was twinned. The twin law was 
> -h,-k,l. So it should be merohedral twinning.
> I read in a paper, that in case of merohedral twinning you cannot see it in 
> the diffraction pattern. But in my case that seemed not to be the case 
> because of splitted reflections. Or am I wrong???
> 
> I would be very happy to hear your opinion on that. Thanks in advance!
> 
> Best regards,
> Marina
> 
> -- 
> Marina Gárdonyi
> 
> PhD Student, Research Group Professor Dr. Klebe
> 
> Department of Pharmaceutical Chemistry
> 
> Philipps-University Marburg
> 
> Marbacher Weg 6, 35032 Marburg, Germany
> 
> Phone: +49 6421 28 21392
> 
> E-Mail: marina@pharmazie.uni-marburg.de
> 
> http://www.agklebe.de/
> 
> 
> 
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Re: [ccp4bb] Student Question--Negative Difference Density in some Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model Structure

[Mark J van Raaij]

Thanks Huw and Ian for shedding light on this.
This is an entry from 2010. If I remember correctly, we submitted the 
uniquefied mtz after truncate to the PDB, which was converted to text format 
(some kind of cif) by the PDB. Not sure if the final format dates from 2010, in 
which case mea culpa for not checking this well enough at the time and not 
knowing enough about mmcif standards. Or if there were later 
conversions/adaptations to the format that introduced these non-standard 
additions of 0 and o?
Could it affect other entries? Ours, but also others?

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 23 Feb 2021, at 13:50, Ian Tickle  wrote:
> 
> 
> Hi Huw
> 
> Yes well spotted.  Wherever I+/sigI+ are missing I/sigI have been set to 
> zero, which means that STARANISO will ignore those reflections; however 
> F/sigF in those cases have been set correctly to F-/sigF-.
> 
> In any case a zero value should not be used to indicate missing datum.  Also 
> I notice that all the test-set flags are set to 'o' (observed).
> 
> Thanks
> 
> -- Ian
> 
> 
> 
> On Mon, 22 Feb 2021 at 21:45, Huw Jenkins 
> <288da93ae744-dmarc-requ...@jiscmail.ac.uk 
> <mailto:288da93ae744-dmarc-requ...@jiscmail.ac.uk>> wrote:
> Hi Gerard,
> 
> > On 22 Feb 2021, at 19:38, Gerard Bricogne  > <mailto:g...@globalphasing.com>> wrote:
> > 
> > Did you perhaps deposit only part of the data you collected for the
> > remote wavelength? For example, only one of several orientations that you
> > might have collected in order to try and fill the cusp? 
> 
> data_r2xgfsf in the 2xgf-sf.cif file from the PDB contains 47650 reflections 
> of which 6932 have:
> 
> I, sigI of 0., 0.
> I(+), sigI(+) listed as missing (? ?) 
> I(-), sigI(-) recorded.
> 
> 47650 - 6932 = 40718 which is the number of reflections listed as the overall 
> total Nref(obs) in the first table in the STARANISO logfile. 47650/47737 (the 
> total for Nref(all) in the same table) is 99.8% complete.
> 
> Is it possible the count of Nref(obs) does not include these 6932 reflections 
> where only I(-) was recorded?
> 
> Best regards,
> 
> 
> Huw
> 
> 
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Re: [ccp4bb] Student Question--Negative Difference Density in some Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model Structure

[Mark J van Raaij]

Hi Gerard,

fortunately I still have the data for this structure on my computer - and not 
on tape or somesuch.
Below is the summary of the SCALA logfile, from which the statistics were 
quoted. Collection was a single sweep of 180 degrees. I'll send you privately 
the complete log file and the output mtz (happy to share with other people 
also, but don't want to post big files to people to the BB in general).
Cell parameters are exactly the same, so I don't think we've submitted the 
wrong mtz to the PDB by mistake.


Summary data for   Project: D9-25 Crystal: nat Dataset: rm

   Overall  InnerShell  OuterShell
  Low resolution limit   22.00 22.00  2.32
  High resolution limit   2.20  6.96  2.20

  Rmerge 0.132 0.034 0.440
  Rmerge in top intensity bin0.052- -
  Rmeas (within I+/I-)   0.182 0.048 0.606
  Rmeas (all I+ & I-)0.177 0.048 0.598
  Rpim (within I+/I-)0.125 0.034 0.414
  Rpim (all I+ & I-) 0.090 0.025 0.305
  Fractional partial bias   -0.030-0.046-0.056
  Total number of observations  179203  5537 26037
  Total number unique47653  1546  6906
  Mean((I)/sd(I))  7.3  15.3   2.8
  Completeness99.9  96.8  99.9
  Multiplicity 3.8   3.6   3.8

  Anomalous completeness  84.5  92.1  80.0
  Anomalous multiplicity   2.0   2.0   2.0
  DelAnom correlation between half-sets -0.090-0.032-0.015
  Mid-Slope of Anom Normal Probability   0.911   - -

Outlier rejection and statistics assume that there is anomalous scattering, ie 
I+ differs from I-

Average unit cell:   157.34   53.98  112.75   90.00  100.43   90.00

Space group: C2

Average mosaicity: 0.68

Minimum and maximum SD correction factors: Fulls   1.04   6.74  Partials   1.63 
 13.05

Dataset: D9-25/nat/rm
 written as averaged data to output file /tmp/mark/D9-25_14_2_mtz.tmp

  Maximum resolution: 2.20A




Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 22 Feb 2021, at 20:38, Gerard Bricogne  wrote:
> 
> Dear Patrick, Mark and colleagues,
> 
> Examining the data associated to PDB entry 2XGF using the PDBpeep
> server, i.e. at 
> 
>  http://staraniso.globalphasing.org/cgi-bin/PDBpeep.cgi?ID=2xgf
> 
> shows a large region (marked up in dark blue) of missing measurements where
> the analysis of the trends in the distribution of the local average I/sig(I)
> would have led to expect statistically significant intensities. Such
> systematic patterns of missing data are never good news when it comes to
> maps.
> 
> This raises a couple of questions for you, Mark:
> 
> 1. this is not the shape of a standard cusp, so would there have been
> features of the crystal morphology that made some regions of reciprocal
> space hard to reach in an experiment, apart from the usual cusp caused by
> the 2-fold axis being too close to the rotation axis? 
> 
> 2. this pattern of missing data makes it rather surprising that the 
> completeness reported in the paper is about 99%, all the way out to the
> outer resolution limit; the STARANISO logfile, accessible at  
> 
>  http://staraniso.globalphasing.org/PDB/2xgf.log
> 
> shows significantly lower values, even for the ellipsoidal completeness, of
> about 0.7 at the highest resolution, for the deposited data.
> 
> Did you perhaps deposit only part of the data you collected for the
> remote wavelength? For example, only one of several orientations that you
> might have collected in order to try and fill the cusp? 
> 
> This may seem a digression from the original question about negative
> difference density, but clearly any systematically missing data can create
> additional artefacts in maps and difference maps, besides those originating
> from chemical or radiation damage related causes.
> 
> 
> With best wishes,
> 
> Claus and Gerard.
> 
> --
> On Mon, Feb 22, 2021 at 07:51:11PM +0100, Mark J van Raaij wrote:
>> Hi Patrick,
>> couldn't see any text in you email, just the subject and the picture, but 
>> 2XGF is our T4 long tail fibre tail tip structure from 2010. 
>> (2XGF stands for "2nd eXtraordinary Great Fibre", at least that's 

Re: [ccp4bb] Student Question--Negative Difference Density in some Histidine side chains in Iron Coordination complex in 2XGF T4 Phage Model Structure

[Mark J van Raaij]

Hi Patrick,
couldn't see any text in you email, just the subject and the picture, but 2XGF 
is our T4 long tail fibre tail tip structure from 2010. 
(2XGF stands for "2nd eXtraordinary Great Fibre", at least that's what I 
*modestly* imagine..).
I think it's probably one or more of: 
- noise (was this contoured at 3sigma? or at a specific e/Å3 level? at the end 
of refinements 3sigma corresponds to less e/Å3 than at the start, or at least 
it should).
- perhaps a small percentage of another metal than iron in the site (Co, Ni, 
Cu, Zn?), it's quite clear most of it was iron though from the X-ray 
fluorescence spectrum and the fact that the MAD phasing worked
- perhaps some radiation damage
- ripple effects around the heavy atom sites
Resolution was 2.2Å and data was 99.9% complete, multiplicity 3.8, more 
statistics here: https://www.pnas.org/content/107/47/20287/tab-figures-data 
<https://www.pnas.org/content/107/47/20287/tab-figures-data> (paper 
https://www.pnas.org/content/107/47/20287). 
We didn't consider lowering the occupancy of the irons, or changing from Fe2+ 
to Fe3+, because that would have meant less electrons, not more as suggested by 
the positive density.
In any case, happy that this thread serves to learn something more from the 
comments by the experts.
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 22 Feb 2021, at 16:16, Patrick Needham  wrote:
> 
> 
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> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> Shot 2021-02-22 at 10.08.36 AM.png>




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[ccp4bb] Fwd: [ccp4bb] Postdoc position, Plant Hormone Transporting Membrane Proteins (Denmark)

[Mark J van Raaij]

in case it might be of interest for someone @CNB:


> Begin forwarded message:
> 
> From: Bjørn Panyella Pedersen 
> Subject: [ccp4bb] Postdoc position, Plant Hormone Transporting Membrane 
> Proteins (Denmark)
> Date: 5 February 2021 at 17:20:38 GMT+1
> To: CCP4BB@JISCMAIL.AC.UK
> Reply-To: Bjørn Panyella Pedersen 
> 
> Dear colleagues,
> I have an open postdoc position funded by the ERC (details below).
> Please pass this along to anyone who might be interested. Thanks!
> /Bjørn
> 
> Postdoctoral position in Structure of Plant Hormone Transporting Membrane 
> Proteins at Aarhus University, Denmark.
> 
> online posting:
> https://tinyurl.com/MBGpostdoc 
> 
> We are looking for a highly skilled and motivated postdoc with an interest in 
> working on plant hormone transporting membrane transporters and preferably 
> with a proven track record in the area of structural and/or functional 
> analysis of membrane proteins and an interest for plant biology.
> 
> Starting date is negotiable. Funding is available through an ERC grant for at 
> least 2 years of employment. An extension is possible for up to a total 
> maximum of 4 years of employment as a postdoc.
> 
> The position:
> The position seeks to strengthen ongoing activities in the laboratory of 
> Bjørn P. Pedersen on structure, related to the function and mechanism of 
> plant hormone-transporting membrane proteins as defined by the ERC project 
> MUM-GROW (pedersenlab.dk ). Exact project will be 
> shaped out in dialogue with top runners. The laboratory's interest is the 
> interplay between structure and function of transmembrane transport processes 
> with a focus on metabolite uptake systems, and the methods uses are primarily 
> crystallography and cryo-EM. The group is part of the Section of Structural 
> Biology at Aarhus University.
> 
> The candidate:
> A successful candidate has a relevant Ph.D. degree and a solid and documented 
> background in structural biology, biochemistry and/or biophysics. Experience 
> with membrane protein expression and purification is favored, and the 
> candidate must demonstrate an ability and interest to work with membrane 
> proteins with a structural aim. Applicants should be ambitious, show strong 
> collaborative skills, and be able to take initiatives and responsibility 
> within the work environment.
> 
> The successful candidate is offered:
> - access to a well-developed research infrastructure.
> - a research climate inviting lively, open and critical discussion within and 
> across different fields of research.
> - a working environment with teamwork, close working relations, network 
> activities among young scientists and social activities.
> - a workplace characterized by professionalism, equality and a healthy 
> work-life balance.
> 
> The city:
> In Aarhus you have easy access to beautiful nature, an exciting culture and 
> city life as well as a safe environment for children - a great place for the 
> whole family. The city of Aarhus has everything you need: exciting national 
> and international jobs, delightful residential areas, a rich cultural life, 
> and beautiful surrounding landscape of woods and coastline that make Aarhus a 
> wonderful place to live and work. See https://international.au.dk/life/ 
>  for details. Aarhus University offers 
> relocation service to international researchers. You can read more about it 
> at https://international.au.dk/life/researcherscomingtoau/ 
> .
> 
> Deadline:
> All applications must be made online (https://tinyurl.com/MBGpostdoc 
> ) and received by March 1st 2021.
> 
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Re: [ccp4bb] (scattering factors) f and f" for Sr Heavy atom

[Mark J van Raaij]

here you can look them up:
http://skuld.bmsc.washington.edu/scatter/AS_periodic.html 
<http://skuld.bmsc.washington.edu/scatter/AS_periodic.html>
(first have to calculate the energy)
but I'm afraid that at that wavelength, below the edge, f' and f" are small, so 
not so likely to give good phase info

9.7 kEV, values of f' = -0.65 and f" = 1.3, if I calculated/looked it up 
correctly

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 22 Jan 2021, at 20:36, rohit kumar  wrote:
> 
> Hello All,
> 
> I have data collected at Wavelength: 1.2782 (For Sr Heavy atom) with a 
> resolution of 1.6 A. I was trying to run Crank in ccp4 for SAD phasing and It 
>  asks me to fill the values of (scattering factors) f and f" for the heavy 
> atom. 
> Can anyone please help with this, how to calculate or where to find these f 
> and f" values for Sr heavy atoms?
> 
> Please let me If you need any information from my side.
> 
> Thank you in advance
> 
> 
> -- 
> Regards
> Dr. Rohit Kumar Singh
> Postdoctoral fellow
> 
> 
> 
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Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Mark J van Raaij]

on the day the news came out, I did wonder if the AlphaFold2 team somehow had 
access to all the preliminary PDB files sent around via Gmail (which belongs to 
the same company), but more as a joke/conspirational thought.
"our" target T1052, was also predicted very well by domains and as a monomer. 
It will be interesting to see how well future iterations of the method can 
assemble the complete protein chain and the complete protein chains into the 
correct heteromer.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 9 Dec 2020, at 10:37, Cedric Govaerts  wrote:
> 
> Dear All
> 
> After about 10 (!) years of (very) hard work we solved the structures of our 
> dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
> anisotropic, experimental phasing, and many long nights with Coot and 
> Buster to achieve model refinement. 
> 
> The experimental structure had a well defined ligand nicely coordinated but 
> also a lipid embedded inside the binding cavity (a complete surprise but 
> biologically relevant) and two detergent molecules well defined 
> (experimental/crystallisation artefact).
> 
> As our paper was accepted basically when CASP organisers were calling for 
> targets I offered my baby to the computing Gods. However we only provided the 
> sequence to CASP, no info regarding any ligand or lipid.
> 
> Less than a month after, the CASP team contacted us and send us the best 
> model.  In fact it was 2 half models as the transporter is a pseudo dimer, 
> with the N-lobe and C-lobe moving relative to each other during transport 
> cycle, thus divided as two domains in CASP.
> 
> The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. 
> And yes, group 427 was the superpower (did not know at the time that it was 
> AlphaFold).
> 
> We had long discussions with the CASP team, as -for us- this almost exact 
> modelling was dream-like (or science fiction) and -at some point- we were 
> even suspecting fraud, as our coordinates had travelled over the internet a 
> few times around when interacting with colleagues.  The organisers reassured 
> us that we were not the only target that had been “nailed” so no reason to 
> suspect any wrongdoing.
> 
> To this day I am still baffled and I would be happy to hear from the 
> community, maybe from some of the CASP participants.
> 
> The target is T024, the “perfect" models are domain-split version (T024-D1 
> and T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
> Deposited PDB is 6T1Z
> 
> Cedric
> 
> PS: I should also note that many other groups performed very well, much 
> better than I would have dreamed, including on the full protein but just not 
> as crazy-good.
> —
> Prof. Cedric Govaerts, Ph.D.
> Universite Libre de Bruxelles
> Campus Plaine. Phone :+32 2 650 53 77
> Building BC, Room 1C4 203
> Boulevard du Triomphe, Acces 2
> 1050 Brussels
> Belgium
> http://govaertslab.ulb.ac.be/ <http://govaertslab.ulb.ac.be/>
> 
> 
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Re: [ccp4bb] protein oligomer

[Mark J van Raaij]

Dear Shijun,
Just a reminder that the pI calculated from the sequence is not necessarily the 
real pI, and sometimes can very quite a bit due to folding and oligomerization 
of the protein. The first protein I ever expressed had a calculated pI of 9.2 
and a real pI of 7.8 (if I remember correctly), so the difference can be quite 
large. You can determine the real pI by iso-electric focussing. 
Best wishes,
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 20 Jul 2020, at 08:38, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:
> 
> Dear:
> 
> The protein was purified in 4 degree, and the expression level is low, so the 
> aggregation is not by high concentration; the buffer pH is 7.5 which is not 
> colse to the PI 8.6. It should be a dimer when function, but it was 
> aggregated when negative staining. Maybe I could try to add arginine when 
> purification, or do mutantions. anyone has website for prediction the 
> mutation sites of protein?
> 
> Thanks!
> 
> Best,
> 
> shijun
> 
> 
> 
> -原始邮件-
> 发件人:"Nikolay Dobrev" 
> 发送时间:2020-07-19 20:29:49 (星期日)
> 收件人: CCP4BB@JISCMAIL.AC.UK
> 抄送: 
> 主题: Re: [ccp4bb] protein oligomer
> 
> It really depends from the nature of the protein and if is 
> oligomerizing/agregating/forming polymers if any of this is reversible.
> On the other side if you are working with one of the fibril forming protein 
> it will require optimizaiton on its on as they will form naturally polymers.
> 
> Do you observed different specises when you analyze your protein by SEC or if 
> you are able to perfom DLS?
> Additional information regarding your protein will be really helpful for more 
> detalied suggestions how to overcome your protein.
> 
> Best,
> Nikolay
> 
> Nikolay Dobrev 
> Scientific Officer, Protein Expression and Purification Core Facility
> EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
> T +49 6221 387 8633 | M +49 173 684 0532
> twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
> Visit www.embl.org/events <http://www.embl.org/events> for a complete list of 
> all EMBL events.
> 
> On 19/07/2020 14:15, S. Mohanty wrote:
>> Keep the protein concentration low during purification steps along with 
>> using other anti-aggregation agent/s. Make sure that the pH at which you are 
>> purifying is not close to the pI of the protein. Until completely purified, 
>> all purification steps should be performed in a cold room if it is a soluble 
>> protein.
>> 
>> Smita 
>> 
>> 
>> On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga  
>> <mailto:sor.dr...@gmail.com> wrote:
>> 
>> 
>> I am not sure what you mean by polymer formation. Presuming that you have 
>> optimized your protein concentration, pH and salt concentration, you could 
>> try arginine as an anti-aggregation agent in your purification (I presume 
>> you do FPLC). Have a look at chaotropic agents used in protein purification, 
>> The answer is generally dependent on the protein/proteins you are trying to 
>> purify and is not necessarily straightforward.
>> 
>> Kinds regards
>> 
>> On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn 
>> <mailto:21620150150...@stu.xmu.edu.cn>> wrote:
>> Dear all:
>> 
>> Any ideas to decrease protein polymer formation? my protein was easy to form 
>> oligomers and precipitation when do purification,I have tried add glycerol 
>> and DTT,both didn't work. Does anyone has experience to avoid it happening. 
>> Thanks!
>> 
>> Best Regards
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
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>> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>-- 
> Nikolay Dobrev 
> Scientific Officer, Protein Expression and Purification Core Facility
> EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
> T +49 6221 387 8633 | M +49 173 684 0532
> twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/u

Re: [ccp4bb] Quote source inquiry

[Mark J van Raaij]

I think most of us are such "excellent" cryo-coolers that for every beamline 
shift we have multiple crystals with ice ring diffraction :-)

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 16 Jul 2020, at 13:25, Harry Powell - CCP4BB 
> <193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi
> 
> Does anyone bother collecting a powder image (e.g. Si powder) these days so 
> they actually have a reference that can be used to check both the wavelength 
> and the beam centre? Or is this considered just something that old folk do?
> 
> Harry
> 
>> On 16 Jul 2020, at 12:19, Gerard DVD Kleywegt  wrote:
>> 
>> There was a case a few years ago (not too many though) where a 1.6 Å 
>> structure had been solved using an incorrect value for the wavelength (~5% 
>> too low, leading to a cell that was slightly too small for its contents to 
>> be comfortable). It was later corrected so we could compare their validation 
>> statistics. Some interesting observations:
>> 
>> - the geometry had been very tightly restrained so that didn't give a clue
>> about the cell error (WhatCheck only suggested a very small change)
>> 
>> - somewhat surprisingly (I thought) the Ramachandran plot did not improve in
>> the correct model (0.3% outliers in the wwPDB validation report), and the
>> sidechain rotamer outliers even got worse (from 1.5 to 2.5 %)
>> 
>> - the map looked surprisingly good for the incorrect cell
>> 
>> - however, RSR-Z told clearly that the map was not good enough for the 
>> claimed
>> resolution - the model had 24% outliers! (3% in the corrected model which
>> still only put it at the ~50th percentile)
>> 
>> - another good indicator was the clashscore (went from 44 to 7)
>> 
>> - the original model did not include an Rfree, but the R-value (>0.3 at 1.6Å
>> resolution) ought to have provided a clue to the crystallographers and
>> reviewers one would think
>> 
>> It would be interesting to see what would happen if the wavelength would be 
>> set 5% too high.
>> 
>> --Gerard
>> 
>> 
>> 
>> On Thu, 16 Jul 2020, Clemens Vonrhein wrote:
>> 
>>> Hi Robbie,
>>> 
>>> On Wed, Jul 15, 2020 at 07:23:15PM +, Robbie Joosten wrote:
>>>> At the same time if you have a a more relaxed approach to restraints
>>>> than you might find systematic deviations in bond lengths. A test
>>>> for that has been in WHAT_CHECK for decades and it actually works
>>>> surprisingly well to detect cell dimension problems.
>>> 
>>> Indeed.
>>> 
>>>> That said, the problem is uncommon now.
>>> 
>>> Not so sure about that: we all rely on an accurate value of the
>>> energy/wavelength from the instrument/beamline - and if that is off
>>> (for whatever reasons) it will result in incorrect cell dimensions and
>>> a systematic deviation from the various restraints.
>>> 
>>> This would even affect the best experiment done on the best crystal
>>> ... so fairly easy to spot at the refinement stage, especially if such
>>> an energy/wavelength offset is constant over a long period of time on
>>> a given instrument. To spot this at the data collection stage one
>>> would hope that at some point a crystal with very pronounced ice-rings
>>> will be looked at properly (and the fact these are not where we expect
>>> them to should cause some head-scratching).
>>> 
>>> Cheers
>>> 
>>> Clemens
>>> 
>>> 
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>> 
>>> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 
>>> list hosted by www.jiscmail.ac.uk, terms & conditions are available at 
>>> https://www.jiscmail.ac.uk/policyandsecurity/
>>> 
>> 
>> 
>> Best wishes,
>> 
>> --Gerard
>> 
>> **
>>  Gerard J. Kleywegt
>> 
>> http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
>> **
>>  The opinions in this message are fictional.

Re: [ccp4bb] Commercial source of His-tagged protein

[Mark J van Raaij]

Dear All,

many thanks to you all for your suggestions and help! I learned about some 
interesting small companies producing proteins and had other useful 
suggestions, some already shared with the list. Another example of the 
wonderful CCP4bb community.
In the end it looks like we will go for His-tagged GFP, because it will have 
specific advantages for the experiments, and someone can produce it locally 
here for what will be a good price (don't have an exact quote yet and won't 
advertise the outfit unless they desire this).

Lesser known (at least not previously known to me) companies that were 
recommended are:
http://www.molirom.com/ <http://www.molirom.com/>
https://www.37cbio.com/ <https://www.37cbio.com/>
https://www.novusbio.com/ <https://www.novusbio.com/>
Acrobiosystems.com <http://acrobiosystems.com/>
(I'm think I can be sure that they are happy to be named)
 
Best wishes,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 2 Jul 2020, at 11:50, Tristan Croll  wrote:
> 
> Thermo Fisher sells His-tagged GFP quite cheaply. 
> https://www.thermofisher.com/order/catalog/product/A42611#/A42611 
> <https://www.thermofisher.com/order/catalog/product/A42611#/A42611>
> Tristan
> 
>  
> 
> 
> On 2020-07-02 10:31, Mark J van Raaij wrote:
> 
>> Dear All,
>>  
>> Wondering if anyone knows of an economical commercial source of a soluble 
>> His-tagged protein. In principle any His-tagged protein would suffice, 
>> because it's for testing coupling to a surface via the His-tag. Asking for 
>> an international collaborator, who don't have access/expertise of a 
>> molecular biology lab (except us :-). 
>> We can and will make the His-tagged proteins of interest for them, but if 
>> there is an economical and reliable commercial source, it might be worth 
>> using that for initial tests.
>>  
>> Best wishes,
>>  
>> Mark
>>  
>> Mark J van Raaij
>> Dpto de Estructura de Macromoleculas
>> Centro Nacional de Biotecnologia - CSIC
>> calle Darwin 3
>> E-28049 Madrid, Spain
>> tel. (+34) 91 585 4616
>> Section Editor Acta Crystallographica F
>> https://journals.iucr.org/f/ <https://journals.iucr.org/f/>
>> 
>> 
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[ccp4bb] Commercial source of His-tagged protein

[Mark J van Raaij]

Dear All,

Wondering if anyone knows of an economical commercial source of a soluble 
His-tagged protein. In principle any His-tagged protein would suffice, because 
it's for testing coupling to a surface via the His-tag. Asking for an 
international collaborator, who don't have access/expertise of a molecular 
biology lab (except us :-). 
We can and will make the His-tagged proteins of interest for them, but if there 
is an economical and reliable commercial source, it might be worth using that 
for initial tests.

Best wishes,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/





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Re: [ccp4bb] not solely pdb issue: need someone to officially settle the pdb dispute

[Mark J van Raaij]

Another problem is that the structure was apparently originally not cited 
properly, and now still cited as work from the lab of B, rather than as work of 
A...

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 21 Aug 2019, at 11:22, Anastassis Perrakis  wrote:
> 
> If the structure has been deposited in the PDB and thus is public ally 
> available, B (or F, G, Φ, Ξ, Δ, Α or whoever else) has every right to use it 
> in a publication. 
> 
> “A” should follow the advice of Frank and do a happy dance for the usefulness 
> of the work, or if not feeling like dancing she/he could follow my advice 
> that will be offered in Greek: «ξυδάκι». 
> 
> Sent from my iPhone
> 
> On 21 Aug 2019, at 11:13, Flemming Goery  <mailto:flemming_go...@hotmail.com>> wrote:
> 
>> Dear all:
>> A has sought a job in the lab of B. B invited A for a interview with a PPT 
>> oral presentation, as requested B has sent the PPT on the structural biology 
>> research of XXX to B by e-mail, and presented in front of A and his 
>> postdoctoral researcher.
>> 
>> After interview, B requested all research documents (including detailed 
>> reports) on XXX to be sent by A to B by e-mail, A sent, including 2 sets of 
>> pdb for the same structure, one set with solvent, one without. A told B all 
>> intellectual property of the Documents and the research belonged to A, based 
>> on the regulation of A's institute.
>> 
>> B sought a referee from A's institute, to someone A did not agree. It seems 
>> the referee told B one set of PDB has been deposited (the one without 
>> solvent)
>> 
>> Then B did not give the offer to A. A joined Institute D, without 
>> independent funding for the writing (in fact, no salary to support this 
>> writing, and no fee for publication of this work).
>> 
>> Several years later, A found B's paper, i.e., the concerned paper published 
>> in Journal C. In the paper, B has used the information from deposited PDB 
>> for 9 times (already a significant paprt of the paper, not to say the 
>> message from the other Documents sent to B by A). In the paper, it write 
>> something like, 'based on our work on the structure of  (folowed by 4 letter 
>> pdb code)', which implied the structure was solved by the authors of the 
>> paper, rather than by A.
>> 
>> A contacted Journal C, Journal C contacted B, B claimed the deposited PDB 
>> was a public domain knowldge. Journal C took the action to add the reference 
>> to the deposited pdb in the paper.
>> 
>> As mentioned, the paper has mentioned and used the message from the 
>> deposited pdb 9 times, and in the paper the reference mark was not added to 
>> the first occurence of the mentioning of the deposited pdb, but added (only 
>> once for the 9 occurences of depositation code) to a paragraph where it can 
>> be concluded that the authors have used the undeposited pdb with the 
>> solvent. In another words, although reference to the deposited pdb was added 
>> by a correction, from where the reference mark was added, it cannot show 
>> they have refered to the cited pdb, not to say the undeposited pdb with 
>> solvent which they used based on the paragraph information.
>> 
>> A's concern was that: A cannot exclude the possibility that the research in 
>> the paper other the part related to PDB, were fabricated, thus A request 
>> paper retraction as the major clain.
>> 
>> If cannot retratcted, A request to be the correspondence author (sometimes 
>> requets co-first author, sometimes request both co-first author and 
>> co-correspondence author), as without A's work (the PPT presentation, 2 sets 
>> of pdb, all documents), the work in the concerned paper cannot be done. A 
>> regard as having contributed to the initiation of the paper, thus A prefer 
>> to be add as a co-correspondence author if appropriate.
>> 
>> First, can the paper deserve a retraction, and second, can B deserve a 
>> co-author?
>> 
>> Flemming
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1>
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Re: [ccp4bb] not solely pdb issue: need someone to officially settle the pdb dispute

[Mark J van Raaij]

From the story, it seems to be a bit more complicated than that, using not only 
a deposited public-domain PDB, but also other data transferred confidentially 
(undeposited pdb and oral and written reports). This does seem unethical to me. 
I have to admit we only have one side of the story though.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 21 Aug 2019, at 11:22, Anastassis Perrakis  wrote:
> 
> If the structure has been deposited in the PDB and thus is public ally 
> available, B (or F, G, Φ, Ξ, Δ, Α or whoever else) has every right to use it 
> in a publication. 
> 
> “A” should follow the advice of Frank and do a happy dance for the usefulness 
> of the work, or if not feeling like dancing she/he could follow my advice 
> that will be offered in Greek: «ξυδάκι». 
> 
> Sent from my iPhone
> 
> On 21 Aug 2019, at 11:13, Flemming Goery  <mailto:flemming_go...@hotmail.com>> wrote:
> 
>> Dear all:
>> A has sought a job in the lab of B. B invited A for a interview with a PPT 
>> oral presentation, as requested B has sent the PPT on the structural biology 
>> research of XXX to B by e-mail, and presented in front of A and his 
>> postdoctoral researcher.
>> 
>> After interview, B requested all research documents (including detailed 
>> reports) on XXX to be sent by A to B by e-mail, A sent, including 2 sets of 
>> pdb for the same structure, one set with solvent, one without. A told B all 
>> intellectual property of the Documents and the research belonged to A, based 
>> on the regulation of A's institute.
>> 
>> B sought a referee from A's institute, to someone A did not agree. It seems 
>> the referee told B one set of PDB has been deposited (the one without 
>> solvent)
>> 
>> Then B did not give the offer to A. A joined Institute D, without 
>> independent funding for the writing (in fact, no salary to support this 
>> writing, and no fee for publication of this work).
>> 
>> Several years later, A found B's paper, i.e., the concerned paper published 
>> in Journal C. In the paper, B has used the information from deposited PDB 
>> for 9 times (already a significant paprt of the paper, not to say the 
>> message from the other Documents sent to B by A). In the paper, it write 
>> something like, 'based on our work on the structure of  (folowed by 4 letter 
>> pdb code)', which implied the structure was solved by the authors of the 
>> paper, rather than by A.
>> 
>> A contacted Journal C, Journal C contacted B, B claimed the deposited PDB 
>> was a public domain knowldge. Journal C took the action to add the reference 
>> to the deposited pdb in the paper.
>> 
>> As mentioned, the paper has mentioned and used the message from the 
>> deposited pdb 9 times, and in the paper the reference mark was not added to 
>> the first occurence of the mentioning of the deposited pdb, but added (only 
>> once for the 9 occurences of depositation code) to a paragraph where it can 
>> be concluded that the authors have used the undeposited pdb with the 
>> solvent. In another words, although reference to the deposited pdb was added 
>> by a correction, from where the reference mark was added, it cannot show 
>> they have refered to the cited pdb, not to say the undeposited pdb with 
>> solvent which they used based on the paragraph information.
>> 
>> A's concern was that: A cannot exclude the possibility that the research in 
>> the paper other the part related to PDB, were fabricated, thus A request 
>> paper retraction as the major clain.
>> 
>> If cannot retratcted, A request to be the correspondence author (sometimes 
>> requets co-first author, sometimes request both co-first author and 
>> co-correspondence author), as without A's work (the PPT presentation, 2 sets 
>> of pdb, all documents), the work in the concerned paper cannot be done. A 
>> regard as having contributed to the initiation of the paper, thus A prefer 
>> to be add as a co-correspondence author if appropriate.
>> 
>> First, can the paper deserve a retraction, and second, can B deserve a 
>> co-author?
>> 
>> Flemming
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1>
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Re: [ccp4bb] need someone officially settle a pdb dispute for a publication

[Mark J van Raaij]

Dear Flemming,

As I understand it (I may be wrong), the final responsible institutions are 
those where the authors work. But as you say, they sometimes don't even reply - 
or they just may be very slow because they want to be really sure before 
committing to any answer.

But the journal has a responsibility also, to retract the paper if there is a 
serious suspicion the data were not obtained ethically. Of course, it may be 
difficult to prove ownership of a pdb file, if both authors claim ownership 
there is not really a way the journal can decide who is right. In my opinion, 
the journal should officially contact the institutions where the authors work 
to try and resolve this. The institutions may take the journal more seriously 
than a single researcher.

A generally respected institution that may advise on authorship disputes is 
COPE, Committee on Publication Ethics: https://publicationethics.org/ 
<https://publicationethics.org/>
May also take a while though...
They have a database with anonymised examples of previously resolved disputes 
that may be helpful - you may find a similar situation on which they have 
"ruled". These are of course not legal rulings, but are considered by their 
members (most respectable journals) as a strong guideline.
This case may have similarities:
https://publicationethics.org/case/claim-stolen-data-and-demand-retractions 
<https://publicationethics.org/case/claim-stolen-data-and-demand-retractions>

Best of luck,

Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 20 Aug 2019, at 17:45, Flemming Goery  wrote:
> 
> Dear All, 
> 
> A and B belong to 2 different institutes. A claimed B has used his pdb for a 
> publication in Journal C. Journal C did not give the retraction, but permit 
> complain related to the journal publication author issue, with the 
> prerequisite journal C did not have the authority on authorship dispute. Then 
> A has e-mailed to the institute head of B with academic misconduct by B as 
> claim, the institute head of B did not give reply.
> 
> In this situation, can A have the journal  authorship  dispute settled by a 
> neutral reviewer (Journal C view: you (A) need to reach out to the 
> institutions that have authority to adjudicate on such matters, as 
> investigation and adjudication on authorship claims falls outside the remit 
> of journal editors. )? Who are qualified as the neutral reviewer so that the 
> review decision can be submitted to Journal C?
> 
> If you believe you are qualified, or you know somebody or some organization 
> qualified, please let me know and I will introduce the issue to you by 
> separate e-mail (it is best not disseminated, am I right?)
> 
> Best regards.
> 
> Flemming
> 
> 
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Re: [ccp4bb] challenges in structural biology

[Mark J van Raaij]

A recent paper in my favourite journal :-) suggests there is no correlation in 
crystallisation conditions even for similar proteins:
http://scripts.iucr.org/cgi-bin/paper?S2053230X19000141 

As you write, surface loops are likely to be different even for similar 
proteins and those are likely to be important for crystal contacts.



> On 23 Jul 2019, at 10:35, melanie.voll...@diamond.ac.uk 
>  wrote:
> 
> I don't think AI will do our job in future as it heavily relies on the 
> crystal structures for training. However, as a community we should embrace 
> this technology/method to help us solve our structures. And why not start 
> with crystallisation? And again the PDB is in a good position here to enforce 
> standards which will pave the way to make use of all the information in the 
> database to train AI. All chemicals must be IUPAC conform an then the PDB can 
> decide about trivial names based on a set of rules for humans (AI doesn't 
> care how you name it as long as it is consistent). No longer those 20 odd 
> names for ammonium sulphate as Janet pointed out years ago.
> 
> 
> And regarding a magic bullet (as in one size fits all), why should a kinase 
> crystallise in the same condition as a polymerase? They do different jobs in 
> a different micro-environment within the cell so their chemical properties 
> will be different. Perhaps there could be some common ground for evolutionary 
> related molecules but a conserved active site doesn't mean a similar surface 
> for crystal contacts which is the key bit in crystallisation, right?
> 
> 


> But as Kay pointed out, crystallisation is a whole field on its own and will 
> go beyond the GRC and the question asked by James.
> 
> 
> M
> 
> 
> From: CCP4 bulletin board  on behalf of Kay Diederichs 
> 
> Sent: 23 July 2019 08:59:10
> To: ccp4bb
> Subject: Re: [ccp4bb] challenges in structural biology
> 
> If you look at the nice figure at the top of the online article, do you 
> believe that this (or rather, the correct) arrangement of domains/ molecules 
> can be predicted from a couple of correlated mutations, and energy 
> minimization? I think AI is a long way from that.  Finding the correct fold 
> of a compact domain, yes I think it's getting there.
> 
> best,
> Kay
> 
> 
> On Tue, 23 Jul 2019 08:28:42 +0530, Nishant Varshney  wrote:
> 
>> What about AI doing our job in the future?
>> 
>> https://www.nature.com/articles/d41586-019-01357-6?utm_source=Nature+Briefing_campaign=4c1d57fdf3-briefing-dy-20190722_medium=email_term=0_c9dfd39373-4c1d57fdf3-44201949
>> 
>> Best Regards
>> Nishant
>> 
>> On Mon, 22 Jul 2019 at 11:30 PM, Sarah Bowman 
>> wrote:
>> 
>>> I'd like to point out that the MAchine Recognition of Crystallization
>>> Outcomes (MARCO) makes a start to 'deep learning applied to crystallization
>>> outcomes', at least in terms of being able to classify drop images
>>> efficiently.
>>> 
>>> 
>>> 
>>> There is obviously more work to be done to correlate these data with
>>> crystallization cocktail components (which Janet and Tom point out the
>>> difficulties with) and positive outcomes.  It seems the first step really
>>> needs to be consistent descriptions and vocabulary - I fully agree with
>>> Janet here!
>>> 
>>> 
>>> 
>>> Reference on MARCO for those interested: Bruno AE, Charbonneau P, Newman
>>> J, Snell EH, So DR, Vanhoucke V, et al. (2018) Classification of
>>> crystallization outcomes using deep convolutional neural networks. PLoS ONE
>>> 13(6): e0198883. https://doi.org/10.1371/journal.pone.0198883
>>> 
>>> 
>>> 
>>> 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 
>>> 
>>> www.getacrystal.org
>>> 
>>> 
>>> 
>>> sbow...@hwi.buffalo.edu
>>> 716-898-8623
>>> 
>>> 
>>> 
>>> 
>>> 
>>> *From: *CCP4 bulletin board  on behalf of Bernhard
>>> Rupp 
>>> *Organization: *k.k. Hofkristallamt
>>> *Reply-To: *"b...@hofkristallamt.org" 
>>> *Date: *Monday, July 22, 2019 at 1:42 PM
>>> *To: *"CCP4BB@JISCMAIL.AC.UK" 
>>> *Subject: *Re: challenges in structural biology
>>> 
>>> 
>>> 
>>> What about 'deep learning' applied to crystallization outcomes? Can it
>>> guide individual trials better than intuition? Can it find previously
>>> unknown promising combinations on a larger scale?
>>> 
>>> 
>>> 
>>> I think several people were well aware of this need for some sort of sound
>>> machine learning already 15 years ago but we had no cloud based AI
>>> 
>>> services thenmaybe it is time to pick this up - particularly if face
>>> recognition can classify the fine detail in 

Re: [ccp4bb] [EXTERNAL] [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

[Mark J van Raaij]

The validation reports are pretty bad, so it seems to be a case where the 
referees and editor have not checked them.
And, like Herman wrote, the models do not appear to agree well enough with the 
maps - in all three cases.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 19 Jul 2019, at 15:46, herman.schreu...@sanofi.com wrote:
> 
> Hi Rhys,
>  
> There is definitively some density present for a ligand, but the active site 
> region looks completely misfitted, and the ligand density may also belong to 
> unfitted protein residues. One first needs to get the protein chain right, 
> and should then look if there would still be density available to fit the 
> ligand.
>  
> Best,
> Herman
>  
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK 
> <mailto:CCP4BB@JISCMAIL.AC.UK>] Im Auftrag von Rhys Grinter
> Gesendet: Freitag, 19. Juli 2019 15:22
> An: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> Betreff: [EXTERNAL] [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
>  
> EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk 
> <mailto:owner-ccp...@jiscmail.ac.uk>
>  
> 
> Hi All,
>  
> I was chatting with a colleague during a recent synchrotron visit and they'd 
> recently come across some ligand/drug bound structures associated with a 
> paper recently published in a high impact factor journal.
>  
> They had pulled the associated SFs from the PDB and found that the electron 
> density associated with these ligands didn't match that reported in the paper 
> and certainly wasn't sufficient to model the alleged ligand.
>  
> I also pulled the structure factors and after refinement in the 
> presence/absence of the alleged ligand I also feel that the density present 
> does not warrant modelling of the ligand. 
>  
> I was hoping that the community might be able to give me an outside opinion 
> on these datasets (PDB IDs: 6MO0, 6MO1, 6MO2) and if the problem associated 
> with the data is verified, provide some advice on how to proceed. 
>  
> This isn't the first occasion I've seen ligand bound structures with 
> questionable density deposited in association with papers in well respected 
> journals. Despite improvements to validation I feel that this problem is 
> widespread.
>  
> Best Regards,
>  
> Rhys
>  
> -- 
> Dr Rhys Grinter
> NHMRC Postdoctoral Researcher
> Monash University
> +61 (0)3 9902 9213
> +61 (0)403 896 767
>  
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] MR for coiled coil structure

[Mark J van Raaij]

This sounds like a case for the Arcimboldo program developed by the Uson group 
(http://chango.ibmb.csic.es/ <http://chango.ibmb.csic.es/>), of which three 
versions are in CCP4 (Borges, Lite and Shredder).
This will use short secondary structure elements in a mixed MR/ab initio 
approach.
We almost never get it to work with our well-diffracting beta-structured 
proteins :-(, but for alpha-helical proteins it apparently has a really good 
success rate.

a recent paper on use of Arcimboldo for coiled coils is here:
https://journals.iucr.org/d/issues/2018/03/00/cb5097/ 
<https://journals.iucr.org/d/issues/2018/03/00/cb5097/>

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 10 Jul 2019, at 10:00, Shengyang Jin  wrote:
> 
> Dear all,
> 
> We recently acquired a data set (2.0 A, P222) for a coiled coil protein 
> (according to Itasser, QUARK, Robetta, and Phaser). 
> Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the 
> protein is quite novel with no apparent homolog in PDB.   
> We tried to to MR with various models (ab initio or homology based) but with 
> little success. 
> 
> We then tried to use AMPLE, but in ccp4 it always returned this error:  
> __main__.py: error: unrecognized arguments: -use_arpwarp True
> (if we untick arpwarp and choose buccaneer instead, it returns -use_arpwarp 
> False)
> 
> Could anyone help?
> 
> Thank you very much.
> 
> 
> Shengyang Jin
> Nanyang Technological University
> Singapore
> 
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Re: [ccp4bb] resolution

[Mark J van Raaij]

looks like a case of the "if Rfree is lower than 0.300 the structure is 
perfect, if Rfree is higher than 0.300 your structure is completely rubbish" 
police striking again (some reviewers are like this). A bit like if p is 
smaller than 0.05 the effect is definitely real and if p is just about 0.05 
there is definitely no effect...
I would include data to 2.4Å. Or perhaps, try steps and look at maps at 2.4, 
2.5, 2.6Å etc. and then decide (PDBredo can do this automagically). And then 
take 2.4Å or some limit much closer to 2.4Å than 3.0Å.
At 2.4Å (or 2.5Å or 2.6Å) there would be much more info and maps should look 
better than at 3.0Å, even if the Rs are a bit higher.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616


> On 5 Jul 2019, at 15:48, Sam Tang  wrote:
> 
> Dear all
> 
> Hello again
> 
> Thanks a lot for the numerous input. 
> 
> I received a dataset which was processed to 2.4A but refined to 3A -- this 
> was the background I raised this question in the first place. Then I looked 
> at the aimless statistics. At 2.4A the high resolution bin CC1/2 0.626, 
> I/sigI 2.0, Completeness 84.6, Multiplicity 1.7 (P1 spacegroup).  I suspect 
> the reason for the refinement resolution limit to be set at 3 A was simply 
> due to better Rw/Rf (0.236/0.294 at 3A; 0.284/0.341 at 2.4A).
> 
> Based on these information am I justified to say that data quality at 2.4 A 
> was suboptimal? In this case do you think refining at a (much) lower 
> resolution is acceptable?
> 
> Best regards
> 
> Sam
> 
> On Fri, 5 Jul 2019 at 13:43, Sam Tang  <mailto:samtys0...@gmail.com>> wrote:
> Hello everyone
> 
> Sorry for a naive question. Is there any circumstances where one may wish to 
> refine to a lower resolution? For example if one has a dataset processed to 2 
> A, is there any good reasons for he/she to refine to only, say 2.5 A?
> 
> Thanks!
> 
> Sam Tang
> 
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Re: [ccp4bb] High Rfree in last Shell

[Mark J van Raaij]

Send your manuscripts to any of the IUCr journals! 
Might still get the occasional referee like that, but the editors should ignore 
those comments.
plus you'd be supporting a scientific society [sorry for the advert...]

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 16 Apr 2019, at 22:29, Diana Tomchick  
> wrote:
> 
> Ah, even though it seems a long time ago, approximately one out of every two 
> referee comments that I receive complain about our modern methods for 
> processing, scaling and reporting our data collection/processing/refinement 
> statistics. I’m afraid it’s an ongoing effort at education.
> 
> Diana
> 
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu <mailto:diana.tomch...@utsouthwestern.edu>
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> 
> On Apr 16, 2019, at 3:09 PM, Frank von Delft  <mailto:frank.vonde...@sgc.ox.ac.uk>> wrote:
> 
> Jan, tell your reviewer to join us all in the 21st century.  
> 
> Diederichs and Karplus, Science, about 2 decades ago.  (Technically, 2012 
> <https://science.sciencemag.org/content/336/6084/1030?sid=8becd183-203a-4c7b-8c8d-815e18e15887>,
>  but it really is a long long time ago now.) 
> 
> 
> On 16/04/2019 18:06, Tim Gruene wrote:
>> Dear Jan,
>> 
>> You statistics look quite solid.
>> 
>> R-factors are not good criteria to judge the resolution cut-off. The 
>> weighting 
>> schemes in refinement programs have much improved since the late 1990s. A 
>> good 
>> starting point to learn more is 
>> Rupp's "Against Method: Table 1 -Cui Bono?", https://doi.org/10.1016/j.str 
>> <https://doi.org/10.1016/j.str>.
>> 2018.04.013
>> 
>> Best regards,
>> Tim
>> 
>> On Tuesday, April 16, 2019 6:57:06 PM CEST Jan van Agthoven wrote:
>>> Hi everyone,
>>> I’m trying to publish two structures at 3.1Å resolution with the following
>>> refinement statistics:
>>> 
>>> Resolution range (Å)   49.2-3.1 
>>> 49.3-3.1 Rfactor (%)   
>>> 24.0 (32.4)  23.4 (32.0) Rfree (%) 
>>> 26.6 (29.2) 
>>> 26.3 (31.6)
>>> 
>>> Data collection
>>> Completeness  100 (100) 
>>>   100 (100)
>>> 
>>> Redundancy6.9 (7.0) 
>>> 6.2 (6.3)
>>> 
>>> Molecules in asymmetric unit  1 
>>> 1
>>> 
>>> Average I/σ 14.1 (1.7)  
>>>  15.3 (2.0)
>>> 
>>> Rmerge (%)  14.9 (100)  
>>> 12.7 (100)
>>> 
>>> Rmeas (%)16.2 (100) 
>>>  13.9 (100)
>>> 
>>> Rsym (%)   6.2 (68.6)   
>>> 5.5 (57.1) Wilson B-factor 
>>>65.662.7
>>> 
>>> I’ve been told that the Rfree factor in the last shell are too high. Does
>>> anyone know how I can improve these Rfree factors other then cutting the
>>> resolution, which already is rather low?
>>> 
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
>>> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1>
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1>
> 
> UT Southwestern 
> 
> Medical Center
> 
> The future of medicine, today.
> 
> 
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Re: [ccp4bb] High Rfree in last Shell

[Mark J van Raaij]

> I’ve been told that the Rfree factor in the last shell are too high. 
> 
that's rubbish. You can not judge the absolute value without looking at the 
data and the model.
If you have a certain amount of disorder, smearing, freezing effects, ice 
rings, and/or diffuse scatter, your Rfree will be higher than average. And it 
may be impossible to model this.
If you have very clean data, your Rfree will be lower than average.

Of course, it may be possible to improve the model, but if you have done 
everything you can to improve it, and perhaps enlisted the advice of an 
experienced macromolecular crystallographer and can just not seem to improve it 
further, it is probably not improvable.
You should also table the CC1/2 overall and for the highest resolution shell to 
make sure the data there is not too noisy.

If the model is as good as you can get it for the data you have, the only 
improvement you could make is collect better data.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 16 Apr 2019, at 18:57, Jan van Agthoven  wrote:
> 
> Hi everyone,
> I’m trying to publish two structures at 3.1Å resolution with the following 
> refinement statistics:
> 
> Resolution range (Å)   49.2-3.1   
>49.3-3.1
> Rfactor (%)24.0 (32.4)
>   23.4 (32.0)
> Rfree (%)  26.6 (29.2)
>   26.3 (31.6)
> 
> Data collection
> Completeness  100 (100)   
>  100 (100)
> 
> Redundancy6.9 (7.0)   
>6.2 (6.3)
> 
> Molecules in asymmetric unit  1   
>1
> 
> Average I/σ 14.1 (1.7)
> 15.3 (2.0)
> 
> Rmerge (%)  14.9 (100)
>12.7 (100)
> 
> Rmeas (%)16.2 (100)   
> 13.9 (100)
> 
> Rsym (%)   6.2 (68.6) 
>5.5 (57.1)
> Wilson B-factor 65.6  
>   62.7
> 
> Does anyone know how I can improve these Rfree factors other then cutting the 
> resolution, which already is rather low?
> 
> 
> 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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Re: [ccp4bb] ORCID being mandatory for PDB depositions

[Mark J van Raaij]

to be honest, I would want to be an author on the paper (especially in a 
"glossy" :-)), but perhaps not on all the database entries...only the ones I 
actively contributed to.
I agree being on the paper involves accepting responsibility for all the work, 
and often it is impossible to really be fully responsible, because it would 
imply checking (i.e. redoing) much of it, but at least in the paper byline it 
should say who did what, giving some "safety" if something turns out wrong with 
the omics or the mice, but the structure(s) being fine.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 9 Apr 2019, at 17:22, Phoebe A. Rice  wrote:
> 
> Very good point, and a good argument against the current trend of 
> publications in the glossies including everything from mice to omics to 
> structure all in one manuscript with one set of authors.  Especially since it 
> is being pointed out more vociferously these days (as it should be) that 
> accepting authorship implies accepting responsibility.
> Phoebe
>  
> From: CCP4 bulletin board  <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Mark J van Raaij 
> mailto:mjvanra...@cnb.csic.es>>
> Reply-To: Mark J van Raaij  <mailto:mjvanra...@cnb.csic.es>>
> Date: Tuesday, April 9, 2019 at 9:32 AM
> To: "CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>" 
> mailto:CCP4BB@JISCMAIL.AC.UK>>
> Subject: Re: [ccp4bb] ORCID being mandatory for PDB depositions
>  
> There is something to be said for not everyone being an author of the PDB 
> entry anyway.
> If I were a non structural biologist who for example had made and tested some 
> essential knock-out mice for the paper, I might prefer not to be an author of 
> the PDB entry in case there was something wrong with it (which I might not 
> necessarily be able to judge).
> Similarly, as a structural biologist I might prefer not to be on the 
> knock-out mice database entry in case there was something wrong or unethical 
> in the way the mice were made...
>  
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> http://wwwuser.cnb.csic.es/~mjvanraaij 
> <http://wwwuser.cnb.csic.es/~mjvanraaij>
> Section Editor of Acta Crystallographica F, Structural Biology Communications
> http://journals.iucr.org/f/ <http://journals.iucr.org/f/>
>  
>> On 9 Apr 2019, at 14:49, Robbie Joosten > <mailto:robbie_joos...@hotmail.com>> wrote:
>>  
>> Than it is easy enough. You mustn't create an ORCid for others, this could 
>> indeed get you into GDPR trouble as you are sharing personal data without 
>> consent.
>>  
>> So that leaves the practical bit. If deposition requires an ORCid and a 
>> collaborator does not have an ORCid despite your requests, then that 
>> collaborator cannot be a depositor. Let the record show that this is not 
>> your fault as your hands are tied here.
>>  
>> HTH,
>> Robbie
>>  
>> On Apr 9, 2019 14:33, V F > <mailto:veronicapfiorent...@gmail.com>> wrote:
>>> On 09/04/2019, Mark J van Raaij >> <mailto:mjvanra...@cnb.csic.es>> wrote: 
>>> > Perhaps the poster is referring to the legality of creating an ORCID on 
>>> > behalf of the collaborator? 
>>> 
>>> Yes that is what I meant. (English not my first language)! 
>>> 
>>> > That is how I interpreted it - but perhaps I over-interpreted... 
>>> > 
>>> > Another thing that came to my mind, not everyone has to be an author of 
>>> > the 
>>> > PDB structure. Often all the authors of the corresponding paper are also 
>>> > on 
>>> > the PDB entry, and there is nothing wrong with that, but if a 
>>> > (non-crystallographer?) collaborator really doesn't want an ORCID, he or 
>>> > she 
>>> > doesn't have to be an author of the PDB entry. 
>>> > 
>>> > 
>>> > Mark J van Raaij 
>>> > Dpto de Estructura de Macromoleculas 
>>> > Centro Nacional de Biotecnologia - CSIC 
>>> > calle Darwin 3 
>>> > E-28049 Madrid, Spain 
>>> > tel. (+34) 91 585 4616 
>>> > http://wwwuser.cnb.csic.es/~mjvanraaij 
>>> > <http://wwwuser.cnb.csic.es/~mjvanraaij> 
>>> > Section Editor of Acta Crystallogra

Re: [ccp4bb] ORCID being mandatory for PDB depositions

[Mark J van Raaij]

There is something to be said for not everyone being an author of the PDB entry 
anyway.
If I were a non structural biologist who for example had made and tested some 
essential knock-out mice for the paper, I might prefer not to be an author of 
the PDB entry in case there was something wrong with it (which I might not 
necessarily be able to judge).
Similarly, as a structural biologist I might prefer not to be on the knock-out 
mice database entry in case there was something wrong or unethical in the way 
the mice were made...

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 9 Apr 2019, at 14:49, Robbie Joosten  wrote:
> 
> Than it is easy enough. You mustn't create an ORCid for others, this could 
> indeed get you into GDPR trouble as you are sharing personal data without 
> consent.
> 
> So that leaves the practical bit. If deposition requires an ORCid and a 
> collaborator does not have an ORCid despite your requests, then that 
> collaborator cannot be a depositor. Let the record show that this is not your 
> fault as your hands are tied here.
> 
> HTH,
> Robbie
> 
> On Apr 9, 2019 14:33, V F  wrote:
> On 09/04/2019, Mark J van Raaij  wrote: 
> > Perhaps the poster is referring to the legality of creating an ORCID on 
> > behalf of the collaborator? 
> 
> Yes that is what I meant. (English not my first language)! 
> 
> > That is how I interpreted it - but perhaps I over-interpreted... 
> > 
> > Another thing that came to my mind, not everyone has to be an author of the 
> > PDB structure. Often all the authors of the corresponding paper are also on 
> > the PDB entry, and there is nothing wrong with that, but if a 
> > (non-crystallographer?) collaborator really doesn't want an ORCID, he or 
> > she 
> > doesn't have to be an author of the PDB entry. 
> > 
> > 
> > Mark J van Raaij 
> > Dpto de Estructura de Macromoleculas 
> > Centro Nacional de Biotecnologia - CSIC 
> > calle Darwin 3 
> > E-28049 Madrid, Spain 
> > tel. (+34) 91 585 4616 
> > http://wwwuser.cnb.csic.es/~mjvanraaij 
> > Section Editor of Acta Crystallographica F, Structural Biology 
> > Communications 
> > http://journals.iucr.org/f/ 
> > 
> > 
> >> On 9 Apr 2019, at 14:20, Anastassis Perrakis  wrote: 
> >> 
> >> I am wondering, are there any arguments that would suggest that ORCID is 
> >> not in line with GDPR requirements? You are disclosing your name etc to 
> >> the PDB anyway, does it matter if its through ORCID or not? 
> >> 
> >> Tassos 
> >> 
> >> 
> >>> On Apr 9, 2019, at 14:04, V F  wrote: 
> >>> 
> >>> Dear all, 
> >>> Did anyone observe that oneDep from EBI made ORCID mandatory for 
> >>> deposition? What am I supposed to do if my collaborators do not want 
> >>> to create ORCID? (especially with GDPR I do not want to create ORCID) 
> >>> Just posting here so that some one will respond? my mails are going to 
> >>> /dev/null? 
> >>> 
> >>> Many thanks, 
> >>> VF 
> >>> 
> >>>  
> >>> 
> >>> To unsubscribe from the CCP4BB list, click the following link: 
> >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> >> 
> >>  
> >> 
> >> To unsubscribe from the CCP4BB list, click the following link: 
> >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> > 
> > 
> >  
> > 
> > To unsubscribe from the CCP4BB list, click the following link: 
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> > 
> 
>  
> 
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Re: [ccp4bb] ORCID being mandatory for PDB depositions

[Mark J van Raaij]

Perhaps the poster is referring to the legality of creating an ORCID on behalf 
of the collaborator?
That is how I interpreted it - but perhaps I over-interpreted...

Another thing that came to my mind, not everyone has to be an author of the PDB 
structure. Often all the authors of the corresponding paper are also on the PDB 
entry, and there is nothing wrong with that, but if a (non-crystallographer?) 
collaborator really doesn't want an ORCID, he or she doesn't have to be an 
author of the PDB entry.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 9 Apr 2019, at 14:20, Anastassis Perrakis  wrote:
> 
> I am wondering, are there any arguments that would suggest that ORCID is not 
> in line with GDPR requirements? You are disclosing your name etc to the PDB 
> anyway, does it matter if its through ORCID or not? 
> 
> Tassos
> 
> 
>> On Apr 9, 2019, at 14:04, V F  wrote:
>> 
>> Dear all,
>> Did anyone observe that oneDep from EBI made ORCID mandatory for
>> deposition? What am I supposed to do if my collaborators do not want
>> to create ORCID? (especially with GDPR I do not want to create ORCID)
>> Just posting here so that some one will respond? my mails are going to
>> /dev/null?
>> 
>> Many thanks,
>> VF
>> 
>> 
>> 
>> 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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Re: [ccp4bb] problem with the symmetry water molecule

[Mark J van Raaij]

and set the occupancy of that water molecule to 0.50

> On 11 Mar 2019, at 17:17, Phil Jeffrey  wrote:
> 
> Hello Firdous
> 
> You are seeing two because you are displaying crystallographic symmetry and 
> you are seeing its symmetry mate.  Coot only places one (check the PDB file) 
> but displays the second generated by symmetry.  It pays to place that water 
> molecule as precisely as possible on the symmetry axis so that refinement 
> programs will treat this as a special position water and eliminate the extra 
> one - i.e. make it as close as possible to its symmetry mate.
> 
> Phil Jeffrey
> Princeton
> 
> On 3/11/19 12:09 PM, Firdous Tarique wrote:
>> Hello everyone
>> I am having a difficult time fitting a water molecule which is right at the 
>> centre of symmetry. Every time I am trying to fit one water molecule it fits 
>> two because of the symmetry atom is at the same place. What is the best way 
>> to solve this problem? I am talking about the water molecule where two 
>> molecules are paced at one place (4th position in the semicircle having both 
>> pink and purple).
>> Thanks
>> Firdous
>> Screen Shot 2019-03-11 at 12.00.21 PM.png
> 
> 
> 
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Re: [ccp4bb] Nitril groups not present on compounds

[Mark J van Raaij]

Hi Maria,

from the picture, it looks like disorder to me. Note that the three non-H atoms 
at the top of the ring also have significantly less density than the five non-H 
atoms that are closer to the protein. Perhaps the compounds is just quite 
wobbly in it's binding site.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 26 Feb 2019, at 13:38, Maria Håkansson  
> wrote:
> 
> Dear all,
> 
> We have experienced nitril groups not present in the electron density
> after data collection on compounds bound to different proteins.
> 
> In one of the projects the compound was tested by MS to check that
> the nitril is present in solution before data collection (crystals used to 
> for MS analysis).
> The nitril should be present since the molecular weight of the compound is 
> the same.
> 
> Also the crystals have been exposed using 10% of the beam (instead of 100%) 
> to try to make 
> the effect of radiation damage as small as possible. Still the nitril is not 
> present.
> 
> In both cases the nitril atoms have been modeled with high temperature 
> factors but
> the electron density is missing, see parts of the compound in the image below 
> contoured at 1 sigma level and in a 1.1 Å electron density map.
> 
> Has anyone else experienced this?
> 
> Best regards,
> Maria
>  
> 
> 
> Maria Håkansson, PhD, Crystallization  13.34.17.png>Facility Manager
> Principal Scientist
> 
> SARomics Biostructures AB
> Medicon Village
> SE-223 81 Lund, Sweden
> 
> Mobile: +46 (0)76 8585706
> Web: www.saromics.com <http://www.saromics.com/>
> 
> 
> 
> 
> 
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Re: [ccp4bb] Live stream of CCP4 Study Weekend?

[Mark J van Raaij]

Good morning and happy 2019,

There was an announcement on Twitter yesterday:
https://twitter.com/ccp4_mx <https://twitter.com/ccp4_mx>
(second post as of writing this)

pointing here: 
https://stfc.ukri.org/about-us/our-purpose-and-priorities/requesting-information-from-uk-research-and-innovation/webinars/

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 9 Jan 2019, at 09:39, Kay Diederichs  
> wrote:
> 
> Good morning everybody,
> 
> I haven't seen any announcement about the streaming of the Study Weekend ... 
> I'd be really interested to watch and listen!
> 
> Any hints?
> 
> thanks,
> 
> Kay
> 
> 
> 
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Re: [ccp4bb] Structure solution - hexapeptide

[Mark J. van Raaij]

When I was at Santiago de Composrela Univ., we have had success with the CCP4 
program ACORN even at around 1.2 Å resolution. Also peptides, no heavy 
atoms.Didn't do this myself though I have to admit, but could put you in 
contact with the people who did.
Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij

Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij
 Original message From: Kristof Van Hecke 
 Date: 02/08/2018  14:53  (GMT+01:00) To: 
CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Structure solution - hexapeptide 
Dear all, 

I’m trying to solve a structure of a (modified) hexapeptide:
- inhouse (very decent) data up to 0.8 Angstrom
- average redundancy = 10 
- according to the Matthews coefficient of 1.88 with 34.77 %solvent, there 
should be 3 Nmol/asym
- ‘large’ unit cell of about a=54, b=54, c=12 
- SG = P3(1)12 or P3(2)12 

As there’s (presumably) only C, H, N and O in the structure, I’m not able to 
solve this via Direct Methods, Charge Flipping etc,. 
Trying MR (with Phaser) doesn’t give any results either, as there’s hardly any 
homologous models


Has anyone encountered a similar problem please, and could provide any possible 
solutions? 
(building in heavy atoms isn’t my first option at the moment,. )


Thank you very much

Regards

Kristof 


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[ccp4bb] Some sort of "closure"?

[Mark J van Raaij]

Not sure if this has been posted here yet, but I don't remember it and only 
just saw this:
https://www.federalregister.gov/documents/2018/04/16/2018-07782/findings-of-research-misconduct
 
<https://www.federalregister.gov/documents/2018/04/16/2018-07782/findings-of-research-misconduct>
[it's about the famous case of fabricated structures and structure factors]

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/





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Re: [ccp4bb] does 12 A diffraction worth optimization

[Mark J van Raaij]

it's always worth trying optimization, you never know.

also try to get a room-temperature diffraction image of your crystal, only then 
will you know if the cryo or freezing didn't damage it. If at RT it diffracts 
to high resolution, you will then know that you have to work on the 
cryo-conditions or on different mounting techniques 
(https://doi.org/10.1107/S0907444911031210 
<https://doi.org/10.1107/S0907444911031210>).

RT diffraction can be done traditionally in a glass capillary, using a plastic 
capillary (like the Mitegen ones: 
https://www.mitegen.com/product/micrort-room-temperature-starter-kits/ 
<https://www.mitegen.com/product/micrort-room-temperature-starter-kits/>), or 
in-plate, like described in this paper for instance: 
https://doi.org/10.1107/S0907444911023249 
<https://doi.org/10.1107/S0907444911023249>.

Yet another option might be controlled dehydration: 
https://pubs.acs.org/doi/10.1021/cg500890r 
<https://pubs.acs.org/doi/10.1021/cg500890r>.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 3 Mar 2018, at 02:34, Natalia O <natalie.c...@gmail.com> wrote:
> 
> Hello,
>  
> I got crystals of protein-nucleic acid complex, rod-shape, reproducible, 
> don’t visibly get damaged upon freezing; however they gave diffraction only 
> to about 12 A. I tried several crystals. My question is whether such crystals 
> worth optimization. Clearly a 4A diffracting crystal could potentially be 
> optimized to 3 – 2.5A, but if the diffraction that I am getting now is 12A it 
> could suggest that the system is so flexible that getting to 3A with this 
> crystal form is not possible at all. I just wonder if there is any statistics 
> or a rule of thumb about what initial diffraction worth optimization?
> 
> Thank you!
> -Natalia
> 

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Mark J. van Raaij]

This one?http://scripts.iucr.org/cgi-bin/paper?SE0260

Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij
 Original message From: Shintaro Aibara 
 Date: 20/11/2017  21:25  (GMT+01:00) To: 
CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [Off-topic] Comparison of the same 
structure built by many people 
Dear All,
Apologies for the slightly off-topic, but I was wondering if anybody knew of a 
paper/textbook where a protein model was built by multiple people (ranging from 
novice to experienced builders) and compared. I believe the conclusion was that 
while the overall trace was broadly correct, experienced builders maintained 
better geometry compared to beginners.
I distinctly remember reading it a couple of years ago but cannot seem to find 
the figure. If anybody knows of this figure/paper/textbook it would be much 
appreciated if you could point me in the direction.
Yours faithfully,
Shintaro

Re: [ccp4bb] doubt regarding MR search model

[Mark J van Raaij]

With Rs of 43/48% I don't think you can be sure that your spacegroup is right.
You should always try all the spacegroup possibilities until you get a solution 
you are sure is right, i.e. that refines to Rs of around 35% or preferably even 
lower.
More so in the case of screw axes, so try P222, P2122, P2212, P2221, P21212, 
P21221, P22121 and P212121. Phaser can do this automatically for you by 
clicking the right box. 
If necessary, then try lower symmetry like P21 and perhaps P1.
Programs like Xanuda can help.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/

> On 19 Sep 2017, at 16:01, Satvik Kumar <kumarsatvi...@gmail.com> wrote:
> 
> Hello,
> 
> Thanks everyone for your explanations.
> 
> I have pasted the pointless output to provide more information.
> Best Solution:  space group P 21 21 21
>  Laue group probability:   0.959
> Systematic absence probability: 0.818
> Total probability: 0.785
> Space group confidence:0.751
> Laue group confidence   0.951
> 
> Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00
> 
> Also based on L-test, pointless says data does not suggest twinning.
> 
> Yes, the R values go down when I refine in both cases. After 20 rounds of 
> restrained refinement using the coordinates generated by monomer as search 
> model, the Rwork and Rfree are 0.43 and 0.48 
> respectively. Refinement using the coordinates generated by using dimer as 
> search model also results in similar R values. I have attached the plots to 
> show that the R values indeed reduce in both cases. 
> 
> Is my space group correct? Do I need to reexamine the space group even though 
> the probability is high?
> 
> If my space group is indeed correct, how do I decide whether to go ahead with 
> the results generated by the monomer search model or the dimer? 
> 
> Please share your thoughts.
> 
> 
> Thanks,
> Satvik
> 
> On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson <eleanor.dod...@york.ac.uk 
> <mailto:eleanor.dod...@york.ac.uk>> wrote:
> You need to provide a bit more information.
> 
> First of all about the data processing..
> 
> Is the space group correct?
> ways of being misled are:
> Non-crystallographic translations with a shift of ~0.5 along an axis - say a. 
>  This will generate absences in the odd h 0 0 reflections and can make the 
> space group appear to be P 21 21 21 whilst it is really P 2 21 21..
> 
> Perfect twinning can have the same effect. In an orthorhombix space group 
> this can usually only occur if two axes have approximately the same length, 
> but the data processing stats can indicate if that is the case.
> 
> Then - re PHASER. The packing rejection criteria may be set too severely - 
> that seems the case for your solution.
> 
> Best check on any MR solution is: does it refine - give it 20 cycles of 
> mindless refinement and see if the R and FreeR go down.
> 
> Then look at the maps and see if there are obvious corrections to be made..
> 
> Eleanor
> 
> On 18 September 2017 at 14:59, Satvik Kumar <kumarsatvi...@gmail.com 
> <mailto:kumarsatvi...@gmail.com>> wrote:
> Dear Crystallographers,
> 
> I am trying to solve a structure in the space group P212121. Based on 
> Matthews coefficient, there are 4 molecules in the asymmetric unit.
> 
> Based on my limited reading about using of Phaser, I understand that a single 
> chain should be used as search model even though many copies are present in 
> asymmetric unit. Am I correct?
> 
> So when I use a single chain as search model and ask Phaser to search for 4 
> molecules, Phaser identifies a single solution with a warning "The top 
> solution from a TF rescoring did not pack" and a warning "Search request 
> requires more scattering than defined in composition. Composition increased 
> to accommodate search components". But the final values reported "PAK=2 
> LLG=1065 TFZ==22.6" indicate that phaser has solved the problem. 
> 
> Can anyone please explain the meaning of the warning.
> 
> When I inspect the arrangement of the chains (attachment), I observe minimal 
> contact between the chains and a large cavity in the center. Can a crystal 
> form this way?
> 
> I have also tried using the dimer as search model and asking phaser to search 
> for 2 molecules. Even in this case, Phaser finds a single solution but the 
> warning and the advisory still appear as before. The numbers reported reduce 
> a bit to "PAK=1 LLG=722 TFZ==29.2".
> 
> Please help me in understanding these results.
> 
> Thanks,
> Satvik
> 
> 
> 
> 
> 

Re: [ccp4bb] Protein rapidly precipitates when off ice

[Mark J van Raaij]

I'd try varying the pH independent of the theoretical pI, sometimes the real pI 
is very different.
(I've worked on a protein with theoretical pI 9.2, real pI determined by 
iso-electric focussing 7.8).

I'd also try limited proteolysis on the milky sample and see if you can 
solubilise it while a sufficiently interesting protein fragment remains.

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

> On 14 Jul 2017, at 08:14, Debanu Das <debanu@gmail.com> wrote:
> 
> Hi,
> 
> I was in Sung-Hou Kim's group when this work below was performed and
> published and I also tried it out on many occasions. Elegant piece of
> work and certainly worth trying.
> 
> Aside from the suggestions of trying different pH and related optimum
> solubility screening and if higher salt and glycerol are not helping
> when off ice, you can consider the following:
> 
> a) Try not concentrating the protein and/or reducing the expression
> levels. Maybe you do not need to have so much protein if it leads to
> relatively rapid precipitation.
> 
> b) Set up some crystallization screens with the protein before
> concentration, especially if the protein is clean enough after Ni-NTA.
> We crystallized many proteins with Ni-NTA followed by tag cleavage,
> and second IMAC
> 
> c) Do a high speed spin of the precipitated sample to remove the
> precipitate, and run on a gel to verify sample, estimate concentration
> and set up crystallization screens on that. This is related to (a) to
> remove excess protein.
> 
> d) set up crystallization screens at 4C immediately or over a few
> hours if stabilized by higher salt/glycerol and maybe the chemicals in
> the crystallization reagents can stabilize the protein.
> 
> e) If it is a nucleic acid binding protein, try complexes with nucleic
> acid added during purification or right after at 4C. Or try protein
> partners or other ligands.
> 
> f) is the protein Cys rich? Can you anticipate/estimate or model
> surface exposed Cys or S-S bonds? Do you have adequate reducing agent
> in the sample?
> 
> g) Lastly, more esoteric stuff like construct and vector optimization,
> mutations, etc.
> 
> I am sure there may be a few other things you can try and there may be
> more suggestions here.
> 
> Best,
> Debanu
> --
> Debanu Das
> 
> On Thu, Jul 13, 2017 at 10:46 PM, Briggs, David C
> <david.bri...@imperial.ac.uk> wrote:
>> Hi Chris,
>> 
>> What is the theoretical pI of your protein? If it is around pH 7.5, you
>> might try gel filtering your protein into a different buffer/pH combination.
>> Try changing by at least 1 pH unit in either direction.
>> 
>> If the pI isn't a problem, then you might try try solubility screening as
>> outlined...
>> 
>> http://scripts.iucr.org/cgi-bin/paper?dz5020
>> 
>> HTH,
>> 
>> Dave
>> 
>> --
>> Dr David C Briggs
>> Hohenester Lab
>> Department of Life Sciences
>> Imperial College London
>> UK
>> http://about.me/david_briggs
>> 
>> 
>> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Chris Fage
>> <fage...@gmail.com>
>> Sent: Thursday, July 13, 2017 11:40:34 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [ccp4bb] Protein rapidly precipitates when off ice
>> 
>> 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] weird diffraction pattern

[Mark J van Raaij]

- which may well be caused by your cryo-protection or flash-cooling procedure.
I'd try to collect a few images at room temperature to see how good the 
crystals can be and if this procedure can be improved.
To prevent overlaps, it may help to find a way to collect the data with the 
crystal rotating around the most problematic cell axis, which tends to be the 
shortest in the crystal. Bent loops might be helpful.

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



> On 13 Jul 2017, at 11:13, Keller, Jacob <kell...@janelia.hhmi.org> wrote:
> 
> You've got multiple lattices--try seeding approaches mentioned in a 
> recent/current thread.
> 
> JPK
> 
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Thursday, July 13, 2017 3:56 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] weird diffraction pattern
> 
> hello everyone, 
> I would like to seek your opinion on my crystal hits. I am working on a 
> helicase 
> 
> of which the native structure is solved and the all solution statistics are 
> 
> fine. I am trying to crystallize and solve the structure of the protein/ssDNA 
> 
> complex. I recently got some hits from commercial screens using sitting drop 
> 
> vapor diffusion. After crystallization optimization, these crystals diffract 
> 
> weakly but to 3.2 Angstroms for the longer exposure time. However, when the 
> 
> crystals rotate between 120 degrees to 180 degrees, the spots become streaky
> 
> (attached), no matter the crystals are hexagonal or flaky. I have tried to 
> 
> determine the structure by molecular replacement method, but the Rwork/Rfree 
> 
> values are huge (above 0.5) and can’t be reduced further. I suspect the 
> 
> obtained crystals quality and resulting processed statistics is the reason 
> for 
> 
> the observed high Rwork/Rfree values. Are there any suggestions?
> 
> All comments will be appreciated!
> 
> Best,
> Chenjun Tang
> 
> 

Re: [ccp4bb] Se-Met and Se-Cys double labelling

[Mark J van Raaij]

If your data is good enough, your SeMets alone might well be enough.
Soaking native crystals in Hg compounds may also work, avoiding SeMet 
altogether. We have had a lot of success with methylmercury chloride binding to 
free Cys. You may have to experiment with different soaking times and protocols.
Finally, don't give up on MR too early, what matters is the structural 
similarity, not directly the sequence identity. We've had success once with 19% 
identity.
Native protein may be much easier to produce than the SeMet and SeMet/SeCys 
versions (and may differ a lot between proteins).

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

> On 21 Jun 2017, at 17:46, Vito Calderone <calder...@cerm.unifi.it> wrote:
> 
> I am working on a protein having 360 residues. In its sequence there are 3
> Met and 5 free Cys.
> I will need MAD to solve the structure since based on the sequence the
> closest homologue has 20% identity匢 suppose MR would be very unlikely to
> work卻o I would like to express a selenium derivative to exploit MAD.
> Looking in the literature 1 Se-Met every 120 residues seems not to comply
> the threshold to get a good anomalous signal. For this reason I would like
> to exploit both Met and Cys so I would have 8 seleniums per 360 residues.
> Could somenone suggest a reference to a protocol to express the double
> mutant protein in NON auxotrophic strains of E. coli which you have
> experienced working efficiently?
> Thanks

Re: [ccp4bb] in crystallo enzymatic activity

[Mark J van Raaij]

I think in his case the WT does not perform the reaction in the crystal, but 
the mutant does.
Haven't heard of examples of this before, but perhaps the WT needs "something" 
to do the reaction, like a conformational change impossible in the crystals, a 
co-factor or something else, perhaps even important for natural regulation.
And the mutant you make somehow lessened this requirement?
Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser <http://www.cnb.csic.es/~mjvanraaij>.cnb.csic.es/~mjvanraaij 
<http://www.cnb.csic.es/~mjvanraaij>



> On 13 Apr 2017, at 17:35, Bonsor, Daniel <dbon...@som.umaryland.edu> wrote:
> 
> Are you using the same columns for purifying both WT and mutant? Could you 
> have the tiniest fraction of WT contaminating your mutant batches which would 
> then turnover you substrate in crystallo over the couple of weeks? You could 
> try washing the columns/systems with NaOH or pepsin to remove WT, or just use 
> separate columns. 
> 
> Dan
> 
> Daniel A Bonsor PhD.
> Sundberg Lab
> Institute of Human Virology
> University of Maryland, Baltimore
> 725 W Lombard Street N370
> Baltimore
> Maryland
> MD 21201
> Tel: (410) 706-7457
> 
> 
> 
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pierre 
> Nioche
> Sent: Thursday, April 13, 2017 11:28 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] in crystallo enzymatic activity
> 
> Dear CCP4bb,
> 
> We work on an enzyme that we crystallized with two substrates bound in the 
> active site (the reaction transform two substrates into two products). We 
> have also the structure with the two products. We are able to see densities 
> for the substrates when we collect data at different time point 
> post-crystallization (days or weeks later). There is no change over time and 
> no in crystallo enzymatic reaction despite the fact that in solution using 
> the same crystallization solution, the reaction occurs readily.
> This is not surprising and there are already many examples in the literature.
> However, when we crystallize a single amino acid variant (mutant within the 
> active site) with the same two substrates, we initially see the substrates 
> but we then observe in crystallo enzymatic activity and formation of the 
> final products over time. This structure is identical to the one determined 
> with the two products co-crystallized with the enzyme. The crystal packing 
> does not seem to be at play here.
> I understand that in crystallo activities are well documented in the 
> literature and can be induced by addition of ligands, X-rays, change in 
> oxidative environment, etc?
> Here, the substrates are present from the beginning of the crystallization 
> experiments with the same concentration. Nothing is added to the crystals 
> later on. Only the time differentiate the two type of crystals: after a 
> couple of weeks, one has the substrates in the active site (wt) while the 
> other has the products (variant).
> 
> Is anyone aware of similar examples where a variant induce in crystallo 
> enzymatic activity without perturbation of the crystal?
> 
> Thanks,
> 
> Pierre
> Dept of Pharmacology, Toxicology and cellular signaling Paris Descartes 
> University

Re: [ccp4bb] waters with positive FoFc peaks?

[Mark J. van Raaij]

Partially occupied Cs+ comes to mind. At lower resolution the difference may be 
more easily fudged away by the refinement lowering the B.
Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij
 Original message From: Andrew Marshall 
 Date: 13/04/2017  07:59  (GMT+01:00) To: 
CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] waters with positive FoFc peaks? 
Hello all,
I have a 1.8A structure (Rfree/Rwork = 20.5/17.4) with 1420 water molecules 
modelled. There are approximately a dozen waters, all well structured with 
hydrogen bonds to protein atoms, with positive difference density (>4sigma, 
sometimes >5) at their centre. The only ions present in my buffer were Cs, Cl 
and a small amount of Na. I thought Cl ions might be a possibility, but many of 
them are in close proximity to acidic residues and/or one-another. It's 
probably worth noting that the same structure solved using data to 2.25A from 
the same crystal at a different wavelength doesn't contain these peaks (the 
offending waters look normal).
Has any come across this before? Thoughts?
Thanks,
Andrew MarshallPhD CandidateLaboratory of Protein CrystallographyDept. of 
Molecular and Cellular BiologySchool of Biological Sciences
The University of Adelaide

Re: [ccp4bb] High Rfree: Phasing issue or partial crystal disorder

[Mark J van Raaij]

Dear Pravin,

we've had a couple of these unfortunately, in our case the only solution has 
been to find alternative crystal forms. Or several crystal forms in which 
different domains are disordered, but allowing to make a composite, complete 
structure for interpretation.
Your N-terminal domain is probably is several alternative positions in 
different copies of the protein in the crystals. This would give rise to 
disordered density, but not complete flexibility, and thus highish Rs.
However, before being sure, you should probably try integrating in lower 
symmetry space-groups, going down to P1 if necessary. If you don't have a 
dataset of 180 or even better 360 degrees perhaps collect it from a fresh 
crystal. And check diffraction images carefully for missed weak spots and a 
possible larger cell.
In this case, I don't think better phases would help, but you never know. I 
would try placing the nearest structural homologue of the N-terminal domain as 
well as you can and see if some density appears, that way you have corrected 
the solvent envelope more or less and this could maybe help a bit.

Greetings,

Mark

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

> On 11 Apr 2017, at 20:54, Pravinkumar Jagtap <pravinja...@gmail.com> wrote:
> 
> Dear All,
> I am stuck with this problem for 2 months and hope you could help.
> 
> We have a 2.1 A dataset for a 380 amino acid long protein. The space group is 
> I4 (single molecule in asymmetric unit, 48% solvent content) and the dataset 
> is quite perfect (no obvious pathologies). The protein itself is organised in 
> 2 lobes (N and C terminal lobes). The sequence identity to nearest homologue 
> structure is 17%.
> 
> We could  get the phases by SeMet SAD phasing (3A resolution dataset, 5 SeMet 
> (excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe and 2 
> partial occupancy (~0.5 each; present on surface) SeMet in N-terminal lobe). 
> Automated  model building (at 2.1 A) yielded nice model for the C-terminal 
> lobe (215 residues)  and manually I could build parts (around 80 residues) of 
> N-terminal lobe with high confidence. In addition we could also build a 
> ligand which is sandwiched between C and N terminal lobe. 
> 
> However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some patchy 
> density left at the N terminal lobe but as it is discontinuous, I cannot 
> build anything in it (except lots of water molecules). In total I am missing 
> around 85 residues. These residues are predicted to be present in secondary 
> structure (and not flexible).
> 
> As I have around 75-80% model built, I would expect that I would have all the 
> phases and  should get nice density for the remaining part. But as I dont see 
> it, could the rest part be flexible? But again, this is not reflected in the 
> R factors (I would then expect low Rfree).
> 
> Could it be that I still lack phases (due to partial occupancy of SeMeth in 
> N-terminal lobe ) and have to try to get them by heavy metal soaking, or 
> there is disorder in the N-terminal lobe? I have also tried solving different 
> datasets for same crystal but this has not been useful.
> 
> Regards,
> Pravin.
> 

Re: [ccp4bb] Some problems in data processing

[Mark J van Raaij]

Hi Gaoyina,

I guess your crystals ended up in slightly different lattices or with slightly 
different ordering during flash-cooling. 
I this case can see two possible solutions:
- collect more complete data from a single crystal, you may have to "sacrifice" 
some resolution by collecting shorter images or attenuating the beam somewhat.
- try to flash-cool the crystals in a more reproducible way, so they are more 
similar to each other.
Another possibility might be that you have too many spot overlaps, in this case 
you may need to collect thinner-sliced images, offset the detector, put the 
detector further away (i.e. lower resolution), or measure a crystal with a 
different orientation (try put the spindle rotation more or less around the 
long cell axis).
In any of these cases I think you will need to collect more data.
With the current data, in Mosflm data processing you may be able to get more 
complete data with the SEPARATION CLOSE option, if you did not invoke this 
already. In other data processing programs there surely are similar options. On 
the other hand, in the automated data processing pipelines that many 
synchrotrons use now they are probably already activated when necessary (at 
least I have rarely been able to improve on automatically processed data when 
doing it myself later...)

Greetings,

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

> On 11 Apr 2017, at 04:46, 高艺娜 <gaoy...@cau.edu.cn> wrote:
> 
> Dear all,
> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000 
> program to meet requirements for data completeness, but the problem is the 
> Rmerge value was too high to go on every time.
> Did anyone have met the same problem and how to solve it? or some tips for 
> solve this problem？
> 
> All comments will be appreciated!
> 
> Best Regards,
> 

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Mark J van Raaij]

Hello Sutapa,

With codon optimisation you would get more and faster expression and likely 
even more insolubility in E. coli (less time for folding). To get the protein 
more soluble perhaps slower production might help. I guess you could try codon 
de-optimisation :-). Cheaper possibilities are growth at lower temperature, 
using a slower-growing E coli strain, using less IPTG or even no IPTG and just 
relying on the leaky expression, but perhaps you have tried all these already.
Or another expression plasmid with tighter control or a different system like 
yeast.
Codon optimisation for insect cells might be worth it if your analysis shows 
that the current sequence is really badly optimised.
Or perhaps your protein needs a specific natural chaperone...it  might be worth 
studying the natural production of your protein more before designing an 
expression strategy.
Without knowing more details about your protein it's hard to give more help - 
you could try to contact other researchers studying similar proteins.

Greetings,

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



> On 03 Apr 2017, at 07:49, Sutapa Chakrabarti <chakr...@zedat.fu-berlin.de> 
> wrote:
> 
> Dear All,
> 
> We’re trying to express and purify a 1000 residue long protein and have run 
> into the problem that it is completely insoluble when expressed in E.coli and 
> is not expressed at all in insect cells. The usual tricks for improving 
> solubility in E.coli, such as addition of GST/MBP tags, optimising expression 
> media and induction conditions and use of different cell strains, have not 
> led to any improvement. 
> 
> We are now looking into ordering a codon-optimised synthetic gene for this 
> protein and are trying to decide whether it would be worthwhile to 
> codon-optimise for expression in E.coli (given that the protein was expressed 
> but not soluble) or if we should attempt baculovirus expression again with a 
> gene that has been codon-optimised for insect cells. 
> 
> My question is:
> has anyone observed an improvement in the solubility of their target protein 
> using a codon optimised gene? 
> 
> I know of several instances where the use of a codon-optimised gene has led 
> to expression where the native gene sequence did not but am unable to find 
> any references for improvement in solubility. Since codon optimisation 
> significantly alters the translation rate of a gene, I believe this should 
> affect solubility as well; but I’d like to know what the community thinks/has 
> observed before I order an exorbitantly priced gene! 
> 
> Thank you in advance,
> Sutapa
> 
> --
> Sutapa Chakrabarti, Ph.D.
> Institute of Chemistry and Biochemistry
> Freie Universität Berlin
> Takustr. 6 
> 14195 Berlin
> Germany
> Phone: +49-(0)30-83875094
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] binding protein to nickel column

[Mark J van Raaij]

Could it be due to the fact that the protein is a histidine kinase - perhaps it 
is interacting with or even modifying its own his-tag?

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

> On 30 Mar 2017, at 21:20, Shubhangi Agarwal <shubhangiagarwal2...@gmail.com> 
> wrote:
> 
> Hello
> 
> I have a 10kda histidine kinase domain protein with a pI of 9.5.
> It has a C-term his tag and despite using different buffers the protein 
> doesnt bind to the nickel cloumn. it comes out in the flow trhough.
> Buffers used- 50mM tris Ph=8, 300mM NaCl
>50mM tris Ph=7, 300mM NaCl
> 50mM  hepes Ph=7.5, 300mM NaCl
> 50mM tris Ph=8, 300mM NaCl, 10% glycerol
> Can someone suggest to get ensure binding of the protein to the nickel-nta 
> column
> 
> Shubhangi
> PhD student 
> under Dr. Jhimli Dasgupta
> St. Xavier's College
> Kolkata
> India

Re: [ccp4bb] Large number of outliers in the dataset

[Mark J van Raaij]

To be really convinced I think you should also compare the maps at 2.6 and 2.3 
Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better, perhaps 
you are adding noise, but the I/sigma and CC1/2 values suggest you aren’t.
Perhaps try 2.5 and 2.4 Å also.
And perhaps remove a well-ordered aa from the input model, refine at different 
resolutions and compare the difference maps for that aa. Or calculate omit maps 
at different resolutions and compare those.

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

> On 29 Mar 2017, at 17:44, Phil Evans <p...@mrc-lmb.cam.ac.uk> wrote:
> 
> It is not clear to me why you believe that cutting the resolution of the data 
> would improve your model (which after all is the aim of refinement). At the 
> edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t seem to 
> be anything wrong with the Wilson plot. Th R-factor will of course be higher 
> if you include more weak data, but minimising R is _not_ the aim of 
> refinement. You should keep all the data
> 
> I don’t know what xtriage means by “large number of outliers”: perhaps 
> someone else can explain
> 
> Phil
> 
> 
> 
>> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira 
>> <juliana.olive...@lnbio.cnpem.br> wrote:
>> 
>> Hello,
>> I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and CC1/2 
>> = 0.779, the summary data is below), but when I perform Xtriage analysis it 
>> says that “There are a large number of outliers in the data”. The space 
>> group is P212121. When I refine the MR solution the Rfree stops around 30% 
>> and it doesn´t decrease (in fact if I continue refining it starts to 
>> increase).
>> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>> 
>> 
>> 
>> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify 
>> about outliers anymore. I could refine the MR solution very well, the final 
>> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a 
>> good structure.
>> I run Zanuda to confirm the space group and it says that the space group 
>> assignment seems to be correct.
>> Do you think that I can improve my structure and solve it at 2.3 Å or 
>> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify 
>> the resolution cut, right? What should I say?
>> Thank you for your help!
>> Regards,
>> Juliana
>> 
>> Summary data:
>> OverallInnerShell  OuterShell
>> Low resolution limit  51.51  51.51   
>> 2.42
>> High resolution limit  2.30 7.27 
>>2.30
>> Rmerge   0.147   
>> 0.054   0.487
>> Rmerge in top intensity bin0.080   - 
>>  -
>> Rmeas (within I+/I-)  0.155   0.057  
>>  0.516
>> Rmeas (all I+ & I-)0.155   0.057 
>>   0.516
>> Rpim (within I+/I-)0.048   0.017 
>>   0.164
>> Rpim (all I+ & I-)  0.048   0.017
>>0.164
>> Fractional partial bias-0.006 -0.003 
>> 0.146
>> Total number of observations83988 2907   
>>  11885
>> Total number unique  8145307 
>>  1167
>> Mean((I)/sd(I))   9.3   23.9 
>> 2.1
>> Mn(I) half-set correlation CC(1/2)0.991   0.998  
>>  0.779
>> Completeness 99.9 99.5   
>>   100.0
>> Multiplicity10.3 9.5 
>>   10.2
>> 
>> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
>> Space group: P212121
>> Average mosaicity: 1.90
>> 
>> 
>> Juliana Ferreira de Oliveira
>> Brazilian Laboratory of Biosciences - LNBio
>> Brazilian Center for Research in Energy and Materials - CNPEM
>> Campinas-SP, Brazil

Re: [ccp4bb] Ramachandran statistics and referee responsibility

[Mark J van Raaij]

I'd expect imposing Ramachandran restraints to lower the Rfree or at least the 
gap between R and Free, otherwise I would not do it. If there were genuine 
Ramachandran outliers, the restrained model might not have these included, 
while these outliers could potentially be very interesting. Of course, if there 
were many, most would probably not be genuine and genuine ones difficult to 
identify.

For your second Q, I’d vote for:

2. authors/depositors have discretion to submit/publish the model they prefer 
so long as refinement protocol is accurately described and weaknesses such as 
poor Ramachandran statistics are evident in the presentation of the data and 
not concealed

But referees should insist on them also providing the data so that whoever 
wants could re-refine the structure to their liking.

Imposing Ramachandran constraints should also be clearly described and not 
concealed…and authors and users of models refined against low-resolution data 
should be realistic about the conclusions one can draw from them.


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

> On 8 Mar 2017, at 15:29, Radisky, Evette S., Ph.D. <radisky.eve...@mayo.edu> 
> wrote:
> 
> Dear all,
>  
> I have two questions.  I would like to find out the community consensus of 
> (1) best practices in refining against  lower resolution data (~4 angstrom) 
> to achieve the best model, and also (2) what manuscript referees should ask 
> for in this regard.  One might encounter a hypothetical situation where 
> standard refinement approaches gave a model with poor Ramachandran 
> statistics.  Imposing Ramachandran restraints gave a model with improved 
> Ramachandran statistics but at the expense of higher Rfree.  I would expect 
> that the model with better geometry is probably more reliable, and wonder if 
> this is the general consensus view?  I also wonder should a referee be the 
> geometry police, or should authors/depositors have discretion to 
> submit/publish the model they prefer so long as refinement protocol is 
> accurately described and weaknesses such as poor Ramachandran statistics are 
> evident in the presentation of the data and not concealed?
>  
> Thanks for your input!
>  
> Evette
>  
> Evette S. Radisky, Ph.D. 
> Associate Professor and Consultant
> Department of Cancer Biology
> Mayo Clinic Cancer Center
> _
> Griffin Cancer Research Building, Rm 310 
> 4500 San Pablo Road 
> Jacksonville, FL 32224 
> (904) 953-6372 
> http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471 
> <http://www.mayo.edu/research/faculty/radisky-evette-s-ph-d/bio-00094471>

Re: [ccp4bb] symmetry and pdb remark

[Mark J van Raaij]

It seems the authors have just chosen a different “parent” molecule to submit 
to the pdb for these two entries, it has nothing to do with the REMARK cards.
It would have been “nicer” if they would have submitted the equivalent “parent” 
molecule, but the unit cells have a relatively big difference anyway 
(81-81-136Å for 2zam; 75-75-132Å for 2zan), so without running a superposition 
the structures don’t superimpose well anyway.
If you want to compare the packing of the two entries in a figure, I would just 
generate the symmetry equivalents in a big enough sphere in Pymol, and then 
keep the 6 equivalent ones of each that you want, un-showing or deleting the 
others by hand.
Note that if you superpose 2zan onto 2zam and then include the CRYST1 card of 
2zam, you are generating the packing for 2zam, not 2zan, so that does not make 
scientific sense.

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

> On 2 Feb 2017, at 04:00, Smith Lee 
> <0459ef8548d5-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Dear All,
> 
> I have a symmetry problem, which I hope I can get your help.
> 
> For both PDB 2zan and 2zam, they are for the same protein, they conformation 
> were similar except that 2zam was apo and 2zam was ATP binding. 2zaz was got 
> by soaking the 2zam crystal with ATP. Both were P65 space group
> 
> However by getting the mates of 2zam and 2zan, you will find their 6 mates 
> arrange differently. Can you explain to me why  their 6 mates arrange 
> differently?
> 
> What is more, by pymol I align 2zan onto 2zam, and I get the PDB for 2zan 
> fitted to 2zam (no any remark information left after pymol saving). Then I 
> add all the remark information from 2zan to the PDB for 2zan fitted to 2zam, 
> and then I view the mates for the remark added PDB for 2zan fitted to 2zam, I 
> find the 6 mates arrange like 2zam, rather like 2zan.
> 
> Thus, will you please explain whi ch remark information decide the mate 
> arrangement, as in the  remark added PDB for 2zan fitted to 2zam? Why after 
> pymol alignment, the same remark information leads to different mates 
> arrangement?
> 
> I am looking forward to getting a reply from you.
> 
> Smith

Re: [ccp4bb] glycosylated density blob?

[Mark J van Raaij]

well yes, looks like a glycosylation to me too.
However, at 2.75Å resolution I think it will be impossible to know which one 
from the density alone.
Perhaps mass spectroscopy can give a clue?
Or get clues from what is known about your protein and your expression system.

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







> On 26 Jan 2017, at 15:45, KH. HEROJIT SINGH, PH D SCHOLAR, PCL 
> <herojitk...@nii.ac.in> wrote:
> 
> Dear All,
> 
>  
> A continuous map from gamma O of serine at expose region was observed. 
> Resolution of dataset is 2.75 A. The serine position is predicted for 
> phosphorylation in-sillico. With fitting with mannose, density is not fully 
> satisfied while with GalNac, some negative density is found. 
> 
>  
> Condition was 15% PEG 3350, Tris 50mM pH 7, 100mM Na2SO4and buffer was  tris 
> 50mM, NaCl 50mM, ph 8.5 , 1mM PMSF. Images from different angle are attached 
> (File size is 300Kb). Arrow in fig pointed the same serine residue.
> 
> 
> Kindly suggest.  
> 
> -- 
> Regards,
> KHUNDRAKPAM HEROJIT 
> Ph D SCHOLAR
> PROTEIN CRYSTALLOGRAPHY LAB.
> NATIONAL INSTITUTE OF IMMUNOLOGY
> NEW DELHI-110067, INDIA

[ccp4bb] PhD fellowships in Spain

[Mark J van Raaij]

Dear future PhD student,

For prospective PhD students there is the current fellowship call open:
https://obrasociallacaixa.org/el/educacion-becas/becas-de-posgrado/inphinit/programme-description
This is a competitive call with good conditions (salary, mobility, 
complementary training), especially for Spanish standards.
If you go to “Search for a Position” and then look for our centre “Centro 
Nacional de Biotecnologia- CNB”, my vacancy on “Structural Biology on 
Bacteriophage Fibres and Tailspikes" turns up.
If you are interested, please apply. There is no need to contact me, because 
the application process selection committee is independent from the proposed 
supervisors. There are also other structural biology projects, and I think 
selected candidates can choose their favourite project based on the final 
shortlist order.
The deadline for application is 2 February, academic records, reference letters 
and a B2 level certificate in english need to be supplied (nationals of 
english-speaking countries are exempted).

Greetings,

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

Re: [ccp4bb] Need suggestion for protein solubility

[Mark J van Raaij]

if you can figure out what is making your protein precipitate, you could put 
something in the collection tubes of the NiNTA column to prevent precipitation. 
For example a bit of concentrated buffer to change the pH, or EDTA.
Another thing, you say your protein is pure after NiNTA, but it may still 
contain some nucleic acids or other impurities not visible on an SDS-PAGE gel 
(a UV spectrum of the protein might give you more information). I would 
recommend dialysis and an ion exchange chromatography second purification step. 
Then you can buffer exchange and concentrate the protein afterwards and 
hopefully it would not precipitate because the non-protein impurities are 
removed.

Mark J van Raaij
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 <http://www.cnb.csic.es/~mjvanraaij>

> On 24 Dec 2016, at 11:52, Praveen Tripathi <tripathipraveen2...@gmail.com> 
> wrote:
> 
> Dear all,
> I am graduate student working on a functional protein which i have cloned in 
> pET-28a vector for recombinant protein production in E.coli expression system.
> The expressed protein is purified on Ni-NTA resins with Imidazole gradient. 
> Surprisingly, i am getting distinct visible white precipitate in pure 
> fractions in eluted fractions itself.
> Please suggest how to make it soluble or how to prevent the precipitation.
> On concentrator the precipitate ration is very much increasing. The protein 
> is pure in soluble as well as precipitate.
> Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer 
> has varying concentration of imidazole varying from 10mM to 300mM.
>  Any kind of suggestion will be highly appreciated.
> My project requires structure determination.
> 
> Thanks in advance.
> 
> Regards
> Praveen

Re: [ccp4bb] R/Rfree values

[Mark J van Raaij]

Dear Rohit,

I wouldn’t judge a structure just by the Rwork and Rfree values, but also by 
the validation and other statistics (bond lengths, angles, Ramachandran plot, 
map quality, fit to map, average B values). If these are all ok, you should be 
able to “get away with” an Rfree of 33%.
In your email you state that you have already made a significant effort in 
different refinement strategies, so perhaps there is no improvement to be made 
there.
The reason for the high-ish Rfree could be that the data is not so good, and 
reprocessing might help. Although the most likely outcome is that you can’t 
significantly improve it, but at least trying will put your mind more at ease.
In the end, the only way to improve the structure, and R-factors, may be to 
grow a better crystal, cryo-protect better and/or collect better data - this 
particular crystal may just have some kind of disorder.

Greetings,

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







> On 14 Dec 2016, at 16:02, rohit kumar <rohit...@gmail.com> wrote:
> 
> Dear All,
> 
> I am solving a data of 2.5 A (C121 space group). Right now the R/Rfree values 
> are 26/33, after many cycles of refinements (With or With out water) the 
> R/Rfree values still same. Zanuda suggests that the space group seems to be 
> correct and the model is looking fine in coot. 
> Some one suggest what is the main problem.
> Should I again process my data? And what is the best way to fine right space 
> group (If problem with space group).
> Please tell me if you need any information regarding may data.  
> 
> -- 
> WITH REGARDS
> Rohit Kumar Singh
> Lab. no. 430,
> P.I. Dr. S. Gourinath,
> School of Life Sciences,
> Jawaharlal Nehru University
> New Delhi -110067

Re: [ccp4bb] Skin on the drop

[Mark J van Raaij]

Microbatch under oil in Terasaki plates or perhaps microdialysis might be an 
option.
Then you don’t have a liquid/air interface. It is harder to do very small 
volumes then, especially with microdialysis.
Or free interface diffusion in thin tubes (or with specialised equipment).

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







> On 17 Nov 2016, at 15:14, xiaoron...@cau.edu.cn <xiaoron...@cau.edu.cn> wrote:
> 
> Dear All,
>  
> How to avoid the skin on the drop? These skins are believed to be a layer of 
> denatured protein or caused by the polyethylene glycol. 
> They will certainly slow down the rate of vapor diffusion. If you have any 
> good suggestions to avoid skin forming? 
>  
> Best wishes,
> Xiaorong Li
> 
> xiaoron...@cau.edu.cn

Re: [ccp4bb] merge different crystal data

[Mark J van Raaij]

it depends a bit on what the reason for the data getting “bad” is.
- if it’s radiation damage, you may need to collect with less intense x-rays or 
take shorter images. Merging isomorphous datasets may also help, but this is 
not always straightforward, for example because different crystals may have 
reacted differently to freezing.
- if the crystal diffracts anisomorphically, i.e. worse in some directions than 
others, than it may be difficult in any case. You may just have to try and grow 
better crystals.
- as the c-axis is much longer than the other two, you may be able to get data 
with less overlaps by making sure your crystal rotation axis is roughly along 
c. You’ll need to figure out where c is with regards to your crystal morphology 
and then mount the crystal in the correct way (a kappa goniometer, or ever 
better, a 4-circle goniometer, will help; if the long axis is along the short 
axis of your crystals, which is observed often, you may need to use bent loops).

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







> On 8 Nov 2016, at 13:45, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:
> 
> Hi ccp4 guys
> 
>  I have a crystal whose spacegroup is I4,and the cell dimension are 
> a=b=83，while ,the C axis is 315.SO the diffraction data is very bad when the 
> crystal started to rotate to some degree ,and it is difficult to process the 
> data .So I wondering there any strategy to resolve this kind of situation ,or 
> any software to merge different crystal data together to process or phasing ? 
>  thanks a lot for your suggestions !!!
> 
>   
>Best Regards
> 
>   
>  SHIJUN 
> 

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Mark J van Raaij]

Hi Vikram,

lots of questions for you:
- what do the images look like? Were there “contaminating” lattices? Did the 
spot shape look ok? Do they get worse later in the data collection, i.e. do you 
have radiation damage? Is the image quality regular or worse at certain angles?
- how good is the data near 2.6Å? Is the data anisotropic?
If the data is not so great, perhaps Rfree 41% is as good as you can get. 
Nevertheless:
- are you sure the molecular replacement solution is correct? Did you see 
density differences for residues you had to “mutate” in COOT that support the 
correctness of the molecular replacement solution?
- what does model validation say? Any regions that still need improving? Any 
major differences between the two monomers that are not explained by the 
density?
- did you try refining with local NCS in REFMAC? If not, this should get the 
Rfree down a bit more.
- residues 1-49 and 370-377 may be genuinely disordered and not modellable, how 
hard did you try?

Hope this helps,

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







> On 25 Oct 2016, at 14:16, Vikram Dalal <vikram.dalal...@gmail.com> wrote:
> 
> Hi everyone,
> 
> We are trying to solve a protein structure of 2.6 A. We have processed it 
> with HKL2000. We have even tried processing with mosflm and xia2. It is in 
> C2221 space group (checked by pointless) and data is not twinned. It has 31% 
> identical with a search model and has 57% sequence coverage. There are 2 
> subunits in asu.
> 
> I did not get proper phases with MOLREP and phaser. I have tried Balbes (R 
> free 50) and Mr BUMP (R free 54).But, density from balbes look some 
> reasonable. 
> So i have refined it in ccp4i and phenix differently and then model build it 
> by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and now i 
> have amino acid 50 to 369 in both chains, still 49 amino acid at N terminal 
> and 8 amino acid at C terminal are missing and some loops are missing in 
> between too. 
> 
> I have tried the ARP/wARP, but it does not work for it. 
> 
> I have even tried phase and build of phenix but condition remain same. i got 
> stuck at R free at 42 and same around 49 amino acids at N terminal and 8 
> amino acids at C terminal and some internal loops still absent.
> 
> Thank you in advance.
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

[Mark J van Raaij]

Summary that I am going to use to justify buying MacOSX instead of Windows (in 
case this is of use to others):

Not available in Windows:
ADXV for looking at crystallographic diffraction images
XDS and HKL2000 for crystallographic data processing
(Auto)SHARP for phasing
ARPWARP for automated model building
(Auto)BUSTER for structure refinement
Uppsala Software Factory programs (Mapman, etc.), manipulation of maps and PDB 
files
Cryo-EM software (EMAN2, BSOFT)
 
Available in Windows but with important limitations:
Phenix (no MR-Rosetta, no parallelization)
DIALS (no parallelization)

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

[ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

[Mark J van Raaij]

Dear All,

our institution requires me to provide a reasoning not to buy a Windows 
computer (I want to buy a new MacOSX system), so I am looking for software that 
does not run or is limited on Windows.

Not available:
(Auto)SHARP
ARPWARP

Available on Windows but with significant limitations
Phenix (no MR-Rosetta, no parallelization)
CCP4 (limitations on file-names)

Please correct me if pertinent and provide additional examples if possible.

Gratefully yours,

Mark

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

Re: [ccp4bb] strange X-ray diffraction diagram――RNA Or Complex？

[Mark J van Raaij]

Dear Yujie,

At first I thought this looked just like diffraction of multiple salt crystals, 
but there are perhaps a couple of rows of regularly spaced spots from a protein 
crystals. However, I think any program will struggle to get anything useful 
from this. 
Perhaps focussing a small beam (ideally microbeam) on different parts of the 
elongated clusters might get more single-crystal diffraction. 
Seeding using these crystal parts may also yield larger single crystals for 
higher resolution single crystal diffraction...

Greetings,

Mark

PS Anyone agree the crystals look like witches brooms? The stick part of the 
broom may be the single crystal.

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







> On 12 Oct 2016, at 08:54, Liu Rachel <liuyujie1...@hotmail.com> wrote:
> 
> 
> Dear everyone:
> 
> Recently, I suffered a problem during my research work. I purified a zinc 
> finger protein, and crystallized as a beautiful cube in a reservoir solution 
> only containing phosphate as the precipitant, no other buffer or molecules. 
> However, regardless of multiple optimization, the crystal diffracted badly 
> (7~8 Å best). I have also tried co-crystallization with dsRNA because this 
> protein can  bind to dsRNA. Then crystals grow in a new condition（2.5M 
> (NH4)2SO4，0.1M BTP,  pH7.0）and its form change to cluster of needle. But the 
> X-ray diffraction diagram is very strange（as shown in the picture）. The Data 
> cannot be processed with HKL2000 either. I want to figure out, could this be 
> a RNA crystal rather than the complex?   Or is there anybody know about the 
> crystal of RNA molecular？
> 
> Thank you very much!
> 
> 
> Yujie Liu 
> Room 2071, research center in life sciences,
> China Agricultural University 
> No. 2 yuanmingyuan west road, Haidian District, Beijing, 100193  P.R. China 
> Tel: (86)-10-62734078
> 
> 

Re: [ccp4bb] Retraction of 2HR0

[Mark J van Raaij]

Gert, 
I think this is a typo in the 3K82 pdb entry, I checked the M section and the 
reference given and the structure used for MR is probably 1BFE (or 1BE9) 
instead of 1BEF.
In any case, if MR works and you rebuild the structure afterwards with an 
automatic building procedure, you might not be aware of the problems of the 
original structure…of course, MR with a made-up structure is not very likely to 
work...
Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij






> On 14 Apr 2016, at 15:48, Gert Vriend <gerrit.vri...@radboudumc.nl> wrote:
> 
> It is funny to see that the structure 3K82 was solved in 2009 (two years
> after the Murthy affair broke) from Murthy's most famous fraudulent
> structure (1BEF). Would be nice to hear which problems they ran into.
> 
> ps 3k82 is just a normal structure that passes most of WHAT_CHECKs tests
> like most other structures do.
> 
> Gert
> Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het 
> handelsregister onder nummer 41055629.
> The Radboud university medical center is listed in the Commercial Register of 
> the Chamber of Commerce under file number 41055629.

Upozornění / Disclaimer <http://www.fzu.cz/e-mail-upozorneni-disclaimer>

[ccp4bb] Easy way to generate symmetry-related protein chains?

[Mark J van Raaij]

Just wondering if there is an easy way to generate symmetry-related chains, 
necessary for instance to join protein chains into the biologically relevant 
multimers.
What I do now is look up the correct symmetry and translation operator in COOT 
or PYMOL and input that in PDBSET, but there may be easier ways.

in the CCP4bb archive I found the following tip for COOT:

Extensions - Modelling - Symm Shift Reference Chain Here.

but that does not appear to be available in COOT, or not anymore.

Mark J van Raaij
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

Re: [ccp4bb] Easy way to generate symmetry-related protein chains? (summary)

[Mark J van Raaij]

It seems the easiest way is to Save Symmetry Coordinates in COOT and then 
assemble the desired chains in a text editor.

 Extensions - Modelling - Symm Shift Reference Chain Here.
is present if you build COOT yourself, but is not in the pre-built releases 
(for now).

Mark J van Raaij
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 22 May 2015, at 14:24, Mark J van Raaij wrote:

 Just wondering if there is an easy way to generate symmetry-related chains, 
 necessary for instance to join protein chains into the biologically relevant 
 multimers.
 What I do now is look up the correct symmetry and translation operator in 
 COOT or PYMOL and input that in PDBSET, but there may be easier ways.
 
 in the CCP4bb archive I found the following tip for COOT:
 
 Extensions - Modelling - Symm Shift Reference Chain Here.
 
 but that does not appear to be available in COOT, or not anymore.
 
 Mark J van Raaij
 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

Re: [ccp4bb] HIS related crystallography issue

[Mark J van Raaij]

yes, at lowish resolution considering potential H-bonds is often the only way 
to orient the His, but also Asn and Gln side-chains correctly. 
Molprobity (http://molprobity.biochem.duke.edu/) and other programs also check 
this for you, suggesting side-chain flips where necessary or advised. And 
Coot has a special side-chain 180º flip function for you to flip them easily.
At higher resolution you can look at the refined B-factors, if the more 
electron-rich atoms (N in His, O in Asn/Gln) have a much higher B than the less 
electron-rich ones (C in His, N in Asn/Gln), this can also mean the side-chain 
needs flipping.
At even higher solution you may see difference density in wrongly oriented 
His/Asn/Gln side-chains.

Mark J van Raaij
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 20 May 2015, at 14:10, Smith Liu wrote:

 Dear All,
  
 Suppose the protein crystal resolution is about 2-3A, then in the map it 
 should be rather difficult to distinguish the C and N in the sidechain of 
 HIS. In this way we may regard the sidechain of HIS is flippable. But suppose 
 in one flipped conformation of the HIS, the free N in the sidechain of HIS 
 can form H-bond with the neighbour H of the OH of the Thr, in the other 
 flipped conformation of the HIS, the free N in the sidechain of HIS cannot 
 form H-bond with the neighbour H of the OH of the Thr (caused by distance 
 issue).
  
 Suppose whether the H-bond forms between the free N in the sidechain of HIS 
 and the neighbour H of the OH of the Thr was very important biologically, in 
 this situation how can we distinguish whether the H-bond forms?
  
 Smith
 
 

Re: [ccp4bb] Online server for generation of topology cartoons

[Mark J van Raaij]

There are different servers and programs which I use as guides (TOPDRAW comes 
to mind), but for publication purposes I always end up drawing topology 
diagrams myself - for what I consider maximum clarity. It takes time, but I 
prefer to show things the way I think is best showing them, using the output 
from one or more of these programs as guide. It is than also easier to change 
them upon suggestions of collaborators or referees.

Mark J van Raaij
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 20 May 2015, at 11:50, Mohammad Khan wrote:

 Dear all,
 
 Is there any good online server for the generation of topology cartoons of 
 proteins, where one can have a clear layout of the various secondary 
 structures? 
 
 I have tried Pro-Origami, but however I am not veru happy with the output. I 
 have used the default options. Maybe if someone can help me out with various 
 options.
 
 I would want to use the output for publication purposes.
 
 Thanks
 
 Mohammad
 
 

Re: [ccp4bb] Protein precipitation

[Mark J van Raaij]

a third way to use the information would be to reclone the part before and/or 
after the protease cleavage site, i.e. you may have identified (a) stable 
domain(s) that may crystallize much better than the full-length protein.

Mark J van Raaij
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 19 May 2015, at 18:48, Fischmann, Thierry wrote:

 The information about of the cleavage site can be used in two ways : possibly 
 identify the protease - as it has already been suggested - or introduce a 
 mutation at the cleavage site which would prevent clipping from occurring.
  
 The caveat of introducing a mutation is that there is always the possibility 
 that the mutated residue plays a key role.
  
 Good luck
 Thierry
  
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe 
 A. Rice
 Sent: Tuesday, May 19, 2015 12:23 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Protein precipitation
  
 For one of our more easily-degraded proteins, we added a mix of protease 
 inhibitors (those expensive tablets you can buy) and also put a drop of EDTA 
 into each tube in the fraction collector so that any contaminating 
 metal-dependent proteases would be knocked out as soon as the protein came 
 off the metal affinity column.  That seemed to help.  
 Also, if you can get your protein to stick to the next column in whatever 
 buffer it comes off the IMAC column in, there's no need to dialyze or 
 concentration between columns - the faster you get it clean, the better.
 Good luck!
  Phoebe Rice
  
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max 
 [m.mic...@fz-juelich.de]
 Sent: Tuesday, May 19, 2015 1:32 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Protein precipitation
 
 Hi Manjula,
 
 I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
 them all) didn't work. The protein degraded after lysis of bacteria within 20 
 min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
 to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
 in icewaterbath). I performed IMAC, IEX and SEC to get rid of the 
 degradation. Finally the Protein was stable for at least one month. 
 You have to take care about temperature induced pH changes. Some buffers 
 change their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can 
 lead to precipitation if you are working close to the pI of your protein.
 
 Maybe you can try to characterize the protease to find a suitable protease 
 inhibitor. Make some massspectrometry with your degraded sample. An other 
 option is to test via westernblot. If you have some C- oder N-terminal 
 monoclonal well characterized antibody. 
 
 Greets Max
 Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula 
 Ramu [manjula@gmail.com]
 Gesendet: Dienstag, 19. Mai 2015 08:05
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: Re: [ccp4bb] Protein precipitation
 
 Thanks all for the suggestions.
 @ Pius,
 I used bacterial expression system.Yes I centrifuged precipitated protein 
 sample and found more amount in soluble form itself, however within a day 
 protein gets degraded. If I have to further purify the protein I have to 
 concentrate the IMAC elutes and concentrate using filters. In this step 
 protein degradation was more and filter used to block and could not complete 
 the process
 .
 Do you use batch method of elution or gradient???
 Also do you add PMSF in the elution buffer???
  
 Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
 tried using 500mM NaCl too but no change was observed.
  
 
 Thanks and Regards,
 Manjula R 
 Research Scholar
 Department of Biophysics 
 National Institute of Mental Health and Neurosciences
 Bengaluru-29, Karnataka
 INDIA
 E-mail: manjula@gmail.com
 Mobile no:+91-9538553356
 http://www.nimhans.kar.nic.in/
  
 On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN bhanu.hydpri...@gmail.com 
 wrote:
 Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
 for IMAC using Ni-NTA. If your protein requires any additional co-factors 
 (like Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.
  
 On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu manjula@gmail.com wrote:
 Hi all,
 I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm 
 NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed 
 affinity purification. Eluted with 200mM imidazole.  While elution I could 
 see slight turbid in eluted protein (I get pure protein, single band on 
 SDS-PAGE). However, when I centrifuged I couldn't see any pellet. After some 
 time(a day) protein got degraded. In other method I tried cation exchange 
 purification of lysate with MES buffer pH 6.5. Here also I saw slight 
 precipitation and later degradation.
 After this again I

Re: [ccp4bb] Gadolinium complexes- phosphate buffer

[Mark J van Raaij]

Hi Isa,

don't discard SeMet too rapidly if there are few Mets, modern beamlines, 
high-redundancy data collection techniques, and processing and phasing programs 
can extract and use small anomalous signal to get structures even if there are 
less SeMets than generally accepted by the rule of thumb. Especially if you 
can rotate around more than one axis or merge data from different crystals. And 
even if the signal is not good enough and you then need additional phasing, the 
SeMet anomalous maps may be useful in tracing.
If you have Cys, try Hg compounds, my favourite is methylmercury chloride. As 
it binds covalently, even if it precipitates in your drop, you may just get 
just enough soluble to react. We usually add a few grains of the mercury 
compound to the reservoir, mix, cover and let equilibrate with the drop for a 
few hours or overnight, then add a ul of the reservoir to the drop and fish 
crystals after different incubation times.

Greetings,

Mark

Mark J van Raaij
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 18 May 2015, at 11:42, isabelle Lucet wrote:

 Hi All,
 
 We recently obtained a native data set to 2.8A. With no molecular replacement 
 available we are now moving to heavy atom (not enough methionine coverage for 
 seleno-met). Unfortunately  crystals grow is in 1.4 Na/K H phosphate pH 8 and 
 we do not have much room for improvement.
 Any literature you could point to or experience with for example gadolinium 
 complexes, co-crystallization, soaking in phosphate buffer? Are we better off 
 just focusing on class B metals?
 
 Thanks for your advise.
 Kind Regards,
 Isa
 
 
 
 __
 The information in this email is confidential and intended solely for the 
 addressee.
 You must not disclose, forward, print or use it without the permission of the 
 sender.
 __

[ccp4bb] Question for those who use AutoSharp via CCP4i on MacOSX Yosemite

[Mark J van Raaij]

Dear All,

when starting CCP4i via the icon, AutoSharp remains greyed out, suggesting it 
is not installed. However, when I start CCP4i in an XQuartz window (i.e. “ccp4i 
”), AutoSharp can be run.
I guess this may be because in first way my “.profile” file is not read, any 
ideas how to fix this?

Greetings,

Mark J van Raaij
mjvanra...@cnb.csic.es mailto:mjvanra...@cnb.csic.es
http://www.cnb.csic.es/~mjvanraaij http://www.cnb.csic.es/~mjvanraaij

Re: [ccp4bb] Maximising anomalous signal in helical/line scan

[Mark J van Raaij]

Hi Matthew,

William seems to want to increase the x-ray intensity gradually during the 
experiment by augmenting the transmission gradually. It that possible? If so, 
would it be advisable?

Mark J van Raaij
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 24 Apr 2015, at 12:45, Matthew BOWLER wrote:

 Dear William,
 at the ESRF we have a Helical characterisation workflow that will calculate a 
 strategy based on the observed diffraction patterns and the distance between 
 2 points selected on a crystal, best wishes, Matt.
 
 
 
 On 2015-04-24 12:00, William Chao wrote:
 Dear all,
 
 Does anyone have experience in maximising anomalous signal during a
 helical/line scan? Most (all?) automated data collection strategies in
 synchrotrons seem to work on a single point of a crystal but don't
 give strategies for line scan. Would it be advisable to increase the
 recommended transmission as radiation is spread along the crystal? If
 the strategy suggests us to use 10% transmission (100% = 2x10^11 ph/s)
 for a 360-degree collection on a single point, to what amount should I
 increase say along a 200 micron scan with a 20 micron beam (crystal
 dimension 200x100x20)?
 
 Thank you very much in advance for the suggestions!
 
 William
 ---
 
 The Francis Crick Institute Limited is a registered charity in
 England and Wales no. 1140062 and a company registered in England and
 Wales no. 06885462, with its registered office at 215 Euston Road,
 London NW1 2BE.
 
 -- 
 Matthew Bowler
 Synchrotron Diffraction Group
 European Molecular Biology Laboratory
 71, avenue des Martyrs
 CS 90181
 F-38042 GRENOBLE Cedex 9
 FRANCE
 France
 ===
 Tel: +33 (0) 4.76.20.76.37
 Fax: +33 (0) 4.76.88.29.04
 
 http://www.embl.fr/
 ===

[ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Mark J van Raaij]

The abstract of the papers says they used MIR.

Mark J van Raaij
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 23 Apr 2015, at 18:57, Todd Jason Green wrote:
 
 My guess is they had the best data they could get, did molecular replacement 
 with the two halves of the repressor and the dna, got a solution and didn't 
 use appropriate restraints in the refinement. Like Phoebe mentioned, we have 
 better tools for this these days.
 
 
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mark J van 
 Raaij [mjvanra...@cnb.csic.es]
 Sent: Thursday, April 23, 2015 11:49 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!
 
 How reliable is too general a question - it depends on what you want to know.
 At 3.9Å they could probably place the phosphate atoms quite well and see the 
 general fold of the protein.
 Finer details will be less reliable, i.e. where the exact side-chains are 
 etc.
 They could probably have forced more amino acids into favourable 
 Ramachandran angles, but would that have made the structure better? Would 
 these favourable angles have been more right? At 3.9Å you can't know for 
 sure.
 Would they have been able to draw more biological conclusions? I'd say not.
 As long as they do not draw more conclusions in the paper than what is 
 supported by the medium-resolution data, the structure provides useful 
 information.
 
 Mark J van Raaij
 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 23 Apr 2015, at 18:03, Misbah ud Din Ahmad wrote:
 
 Dear crystallographers,
 
 The PDB entry
 http://www.rcsb.org/pdb/explore.do?structureId=3BDN
 has 16.5% Ramachandran outliers. When I opened this PDB file in coot and 
 checked for Ramachandran outliers, the results are:
 In preffered region: 58.04%
 In allowed regions: 19.78%
 Outliers: 22.17%  !
 
 With an R-free of 37.4% at 3.9 A resolution, could you please tell me how 
 reliable this structure of Lambda repressor bound to DNA is?
 
 
 Thanks
 Misbha
 
 
 
 
 
 
 
 

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Mark J van Raaij]

The abstract of the papers says they used MIR.

Mark J van Raaij
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 23 Apr 2015, at 18:57, Todd Jason Green wrote:

 My guess is they had the best data they could get, did molecular replacement 
 with the two halves of the repressor and the dna, got a solution and didn't 
 use appropriate restraints in the refinement. Like Phoebe mentioned, we have 
 better tools for this these days.
 
 
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mark J van 
 Raaij [mjvanra...@cnb.csic.es]
 Sent: Thursday, April 23, 2015 11:49 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!
 
 How reliable is too general a question - it depends on what you want to know.
 At 3.9Å they could probably place the phosphate atoms quite well and see the 
 general fold of the protein.
 Finer details will be less reliable, i.e. where the exact side-chains are etc.
 They could probably have forced more amino acids into favourable Ramachandran 
 angles, but would that have made the structure better? Would these 
 favourable angles have been more right? At 3.9Å you can't know for sure.
 Would they have been able to draw more biological conclusions? I'd say not.
 As long as they do not draw more conclusions in the paper than what is 
 supported by the medium-resolution data, the structure provides useful 
 information.
 
 Mark J van Raaij
 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 23 Apr 2015, at 18:03, Misbah ud Din Ahmad wrote:
 
 Dear crystallographers,
 
 The PDB entry
 http://www.rcsb.org/pdb/explore.do?structureId=3BDN
 has 16.5% Ramachandran outliers. When I opened this PDB file in coot and 
 checked for Ramachandran outliers, the results are:
 In preffered region: 58.04%
 In allowed regions: 19.78%
 Outliers: 22.17%  !
 
 With an R-free of 37.4% at 3.9 A resolution, could you please tell me how 
 reliable this structure of Lambda repressor bound to DNA is?
 
 
 Thanks
 Misbha
 
 
 
 
 
 
 

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Mark J van Raaij]

How reliable is too general a question - it depends on what you want to know.
At 3.9Å they could probably place the phosphate atoms quite well and see the 
general fold of the protein.
Finer details will be less reliable, i.e. where the exact side-chains are etc.
They could probably have forced more amino acids into favourable Ramachandran 
angles, but would that have made the structure better? Would these favourable 
angles have been more right? At 3.9Å you can't know for sure.
Would they have been able to draw more biological conclusions? I'd say not. 
As long as they do not draw more conclusions in the paper than what is 
supported by the medium-resolution data, the structure provides useful 
information.

Mark J van Raaij
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 23 Apr 2015, at 18:03, Misbah ud Din Ahmad wrote:

 Dear crystallographers, 
 
 The PDB entry  
 http://www.rcsb.org/pdb/explore.do?structureId=3BDN
 has 16.5% Ramachandran outliers. When I opened this PDB file in coot and 
 checked for Ramachandran outliers, the results are:
 In preffered region: 58.04%
 In allowed regions: 19.78%
 Outliers: 22.17%  !
 
 With an R-free of 37.4% at 3.9 A resolution, could you please tell me how 
 reliable this structure of Lambda repressor bound to DNA is?
 
 
 Thanks
 Misbha
 
 
 
   
   
 
 

[ccp4bb] CrystalClear tape rolls

[Mark J van Raaij]

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

Re: [ccp4bb] CrystalClear tape rolls

[Mark J van Raaij]

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] Balbes

[Mark J van Raaij]

Just use Amore, Molrep or Phaser and give them the apo structure as model. I 
especially recommend Amore for first-time MRers, as it forces you to understand 
some basics of MR. And it runs very fast.
Molrep and especially Phaser can probably solve more difficult problems with 
lower homology models and are easier to use than Amore, but usually take a bit 
longer.
Balbes is overkill for a problem like this.

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 27 Nov 2014, at 21:19, Atul Kumar wrote:

 Hi all,
 
 I am trying to solve complex structure of my proteins. Apo protein structure 
 is already known. Balbes should take this structure as template for MR, 
 whereas, it is taking some random proteins as template. I was wondering, 
 whether it is synchronized with pdb or uses its own databank. Since structure 
 of this protein deposited in pdb last year, wondering whether or not it is 
 updated  in its local databank, in case it is not synchronized with pdb. Can 
 somebody help to figure out this problem?
 
 regards
 
 Atul 

Re: [ccp4bb] Merge PDB chains

[Mark J van Raaij]

Dear Lu,

- you could just edit the pdb-file by hand in a suitable texteditor (which is 
what I usually do, in MacOSX TextEdit).
- smarter people may find a functionality in COOT, PDBSET or other existing 
program that can do it.
- even smarter people would probably write a script or little program to do it, 
or already have one lying around...

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







On 23 Oct 2014, at 12:51, luzuok wrote:

 Dear all,
Sorry to ask a simple question. There are many SO4 in my PDB file, one 
 belongs to a different chain. I want to merge them into one chain, can anyone 
 tell me how to do this?
 
 Best regards!
 
 Lu Zuokun
 
 
 
 
 --
 卢作焜
 南开大学新生物站A202
 
 

[ccp4bb]

[Mark J van Raaij]

Hi Rohit,

you should really ask Phenix questions on the Phenix bulletin board...
The output means that the molecules found do not pack well in the unit cell and 
overlap more than a certain threshold.
I would first rerun the program with the packing test switched off and look at 
the top solutions. If one looks alright and the overlap is just caused by some 
surface loop(s) in your search model that may have another position in your 
protein, you can just rebuild and go ahead from there. If the overlap is more 
serious, you can look for fewer copies or look for domains separately, if 
appropriate. Or increase the allowed overlap.

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 27 Sep 2014, at 13:34, rohit kumar wrote:

 Dear All,
 
 I am trying to solve a data of 2.9 A resolution in phenix.
 But every time got this error however phenix still give a output PDB. 
 Can any one tell me what is the problem or any solution to overcome this 
 error.
 
 
 
 
 $TEXT:Warning: $$ Baubles Markup $$
 -
 Solutions with Z-scores greater than 13.9 (the threshold indicating a 
 definite solution) were rejected for failing packing test
 #1 TFZ=18.5 PAK=42
 #2 TFZ=18.2 PAK=38
 #3 TFZ=16.3 PAK=80
 #4 TFZ=15.6 PAK=88
 -
 $$
 
 -- 
 WITH REGARDS
 Rohit Kumar Singh
 Lab. no. 430,
 P.I. Dr. S. Gourinath,
 School of Life Sciences,
 Jawaharlal Nehru University
 New Delhi -110067

Re: [ccp4bb] Enigmatic electron density attached to Cys residue

[Mark J van Raaij]

What odds to you give us?
...I bet on disordered DTT covalently bound to the protein.

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 19 Aug 2014, at 16:12, Bernhard Loll wrote:

 Dear all,
  
 We are currently working on a small GTPase. The structure has been solved to 
 1.4 A with two molecules in the ASU. In the difference electron density we 
 can clearly see difference density (in one monomer) attached to a Cys residue.
  
 The protein has been expressed in E. coli. For crystallization experiments 
 the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES 
 pH 7.5 and 50 mM NaCl. Prior to crystallization
 the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM 
 NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol.
  
 The protein crystallized under the following conditions:
 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0
  
 The anomalous signal is too weak to judge the positions of the sulphur atoms. 
 We have performed MS analysis on the protein before crystallization and on 
 dissolved protein crystals. MS revealed a mass difference of about 135 Da, 
 indicating that some chemistry must have went on in the crystallization drop.
  
 The extra electron density has a planar shape and is quite symmetric. We have 
 placed some dummy water molecules in the density. Distances are given in A in 
 the PNG file.
  
 Attached files
  
 coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron 
 density map (blue) @ sigma=+3 after phenix.refine
 coot2.png: same electron densities as in coot1.png, with dummy atoms placed
 coot3.png: same electron densities as in coot1.png, side view
  
 Thanks for your time and efforts.
  
 Cheers,
  
 Bernhard
 -- 
 Dr. Bernhard Loll
 Freie Universitaet Berlin
 Fachbereich Biologie, Chemie, Pharmazie
 Institut fuer Chemie und Biochemie
 AG Strukturbiochemie
 Takustr. 6
 D-14195 Berlin
 Germany
 
 Phone: +49 (0) 30 838-57348
 Fax:   +49 (0) 30 838-457348
 Email: 
 l...@chemie.fu-berlin.de
 
 Homepage: 
 http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/
 coot1.pngcoot2.pngcoot3.png