Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era?

[Javier Gonzalez]

Thank you all for your thoughtful answers.

I'll stick to the crystallographic data and model as much as I can with
reasonable occupancy and B factors, even if the result is a truncated model.

I don't believe in the zero occupancy trick either. I just thought the PDB
was more flexible these days, since the community of structural biologists
seems to have accepted the highly accurate and highly imprecise coordinates
of AF-generated noodle-like loops in the UniProt database.

It sounds like the PDB-DEV database is the right place to deposit those
hybrid models I had in mind, and leave the genuine crystallographic models
for the PDB.

Best wishes,
Javier

On Sun, Jul 28, 2024 at 2:52 PM Guillaume Gaullier <
guillaume.gaull...@kemi.uu.se> wrote:

> Hello Javier,
>
> Placing atoms implies that you know they are present somewhere (possibly
> with some uncertainty on exactly where), but setting their occupancies to
> zero implies that you know they are nowhere at all. This is a paradox.
>
> I think atoms with zero occupancy make no sense in a final deposited model
> (they could be useful as a working intermediate to exclude the bulk solvent
> model, but this is unrelated to what you describe).
> So in this particular case, partial occupancies only make sense for
> multiple conformations (and should add up to 1), as Pavel describes.
>
> If you can’t resolve more than one conformation, maybe a better approach
> is to fix the coordinates to what AlphaFold suggested (which is often
> reasonable, but check their pLDDT to assess this) and refine to let the
> B-factors of these atoms rise. This will convey the large uncertainty on
> their positions. I think it is a valid approach because you know these
> residues are there somewhere (in other words, to me you would need evidence
> of their absence to justify truncating these loops: SDS-PAGE showing that
> the protein is cleaved, for example).
>
> I hope this helps,
>
> Guillaume
>
> On 28 Jul 2024, at 17:32, Pavel Afonine  wrote:
>
> 
>
> Javier,
>
> Flexible loops may be better modeled with ensembles of N models, meaning
> the occupancy of each-one would be 1/N, and the map contours to visualize
> them should be chosen as 1/N sigma (not 1 sigma). While model prediction
> tools such as AlphaFold are helpful, they don't suddenly lift the
> requirement for the atomic model you release to the world to fit the
> experimental data! With this premise in mind, the approaches to validate
> your model geometry and model-to-data fit quality have not changed before
> and after the AlphaFold era.
>
> Whether you truncate residue side chains/loops that you don't see or keep
> them with zero occupancy is a perennial question on this list that has been
> coming up for decades, and I have yet to see an answer that everyone agrees
> on!
>
> All the best,
> Pavel
>
>
> On Sun, Jul 28, 2024 at 8:13 AM Javier Gonzalez  wrote:
>
>>
>>
>> Dear CCP4bb,
>>
>> I'm refining the ~3A crystal structure of a big protein, largely composed
>> of alpha helices connected by poorly-resolved loops.
>> In the old pre-AlphaFold (AF) days I used to simply remove those
>> loops/regions with too high B factors, because there was little to none
>> density at 1 sigma in a 2Fo-Fc map.
>> However, considering that the quality of a readily-computable AF model is
>> comparable to a 3A experimental structure, and that the UniProt database is
>> flooded with noodle-like AF models, I was considering depositing a combined
>> model in the PDB.
>> Once R/Rfree reach a minimum for the model truncated in poorly resolved
>> loops, I would calculate an augmented model with AF calculated missing
>> regions (provided they have an acceptable pLDDT value), assign them zero
>> occupancy, and run only one cycle of refinement to calculate the formal
>> refinement statistics.
>> Would that be acceptable? Has anyone tried a similar approach?
>> I'd rather do that instead of depositing a counterintuitive model with
>> truncated regions that few people would find useful!!
>>
>> Thank you for your comments,
>>
>> Javier
>>
>> --
>> Dr. Javier M. González
>> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
>> Universidad Nacional de Santiago del Estero (UNSE)
>> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
>> Santiago del Estero. Argentina
>> Tel: +54-(0385)-4238352
>> Email  Twitter <https://twitter.com/_biojmg>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>
> -

[ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era?

[Javier Gonzalez]

Dear CCP4bb,

I'm refining the ~3A crystal structure of a big protein, largely composed
of alpha helices connected by poorly-resolved loops.
In the old pre-AlphaFold (AF) days I used to simply remove those
loops/regions with too high B factors, because there was little to none
density at 1 sigma in a 2Fo-Fc map.
However, considering that the quality of a readily-computable AF model is
comparable to a 3A experimental structure, and that the UniProt database is
flooded with noodle-like AF models, I was considering depositing a combined
model in the PDB.
Once R/Rfree reach a minimum for the model truncated in poorly resolved
loops, I would calculate an augmented model with AF calculated missing
regions (provided they have an acceptable pLDDT value), assign them zero
occupancy, and run only one cycle of refinement to calculate the formal
refinement statistics.
Would that be acceptable? Has anyone tried a similar approach?
I'd rather do that instead of depositing a counterintuitive model with
truncated regions that few people would find useful!!

Thank you for your comments,

Javier

-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  Twitter 



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[ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

[Javier Gonzalez]

Dear all,
I'm sure this issue comes up very often, but for the first time in our lab
we need to recover a pET-type expression plasmid from a BL21-like E. coli
strain (NEB's T7 Express).
I know a RecA+ strain is not suitable for plasmid production, but the basic
plan is to grow and mini-prep the cells to recover the plasmid and later
transform another E coli strain (Top10) to make frozen stocks and for
plasmid production.
Is this a regular practice or is there any known protocol we should follow?
Any advice will be greatly appreciated.

Best wishes,
Javier

-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  Twitter 



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Re: [ccp4bb] Validation of structure prediction

[Javier Gonzalez]

Hello,
I think it is perfectly "valid" to run AlphaFold2 / RosettaFold / etc.
models through geometry validation servers (aside from reporting their
intrinsic quality indicators like TM-score and pLDDT distribution, as
mentioned above by Randy Read). Since most experimental structures have a
small percent of Ramachandran outlier residues, a good model ought to have
some too. I´ve run into crystal structures that display Ramachandran
outliers very well supported by the electron density, and that "strain" in
the backbone later on turned out to be favorable for the catalytic
mechanism, and so on... Therefore, if AF2 or RosettaFold algorithms are as
good as they are advertised, they should be able to capture and predict
those outliers too... and it would be very interesting to find out if they
do! A model with a perfect Ramachandran plot is probably as reliable as a
low resolution structure resulting from a heavily restrained geometry
refinement.
By the way this is true only for the polypeptide backbone conformation, I
don't think any algorithm can predict side chain conformations yet (indeed
AF2 offers an optional sidechain energy minimisation to tidy up side chains
in the final models, a least in ColabFold by Sergey Ovchinnikov).
Happy holidays,
Javier

On Tue, Dec 21, 2021 at 10:29 AM Reza Khayat  wrote:

> Hi,
>
>
> Thank you for the help. I've addressed some of the concerns raised here in
> another thread. "Validation" referred to checking geometric parameters;
> however, outstanding geometric parameters do not indicate a structure that
> is comparable to an experimentally determined structure. The structures
> were predicted with the Robetta server and all have, as expected, geometry
> better than most experimental structures.
>
>
> Best wishes,
> Reza
>
>
> Reza Khayat, PhD
> Associate Professor
> City College of New York
> Department of Chemistry and Biochemistry
> New York, NY 10031
> --
> *From:* CCP4 bulletin board  on behalf of Krieger,
> James M 
> *Sent:* Tuesday, December 21, 2021 7:14 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [EXTERNAL] Re: [ccp4bb] Validation of structure prediction
>
> There are also dedicated homology modelling validation tools such as
> ANOLEA (ANOLEA (Atomic Non-Local Environment Assessment) (melolab.org)
> 
> ).
>
> Best wishes
> James
> --
> *From:* CCP4 bulletin board  on behalf of Nicholas
> Clark 
> *Sent:* Tuesday, December 21, 2021 11:57 AM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* Re: [ccp4bb] Validation of structure prediction
>
> Reza,
>
> Thus far, it seems we’ve all assumed this was an AlphaFold or RobettaFold
> model. If this is not indeed the case, it may be worthwhile to “validate”
> your mode by running your sequence through one of these two and using the
> validation from them.
>
> The AlphaFold DB can be found here, with a number of predicted structures:
>
> https://alphafold.ebi.ac.uk
> 
>
> The AlphaFold colab can be found here, although the prediction is not as
> good as AlphaFold 2, as it does not use templates:
>
>
> https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb
> 
>
>
> RobettaFold can be found here:
> https://robetta.bakerlab.org
> 

[ccp4bb] [just for fun] please vote #JBCMethodsMadness #TeamXRC

[Javier Gonzalez]

just one hour left !!
https://twitter.com/jbiolchem/status/1374030429729271811?s=20

-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Javier Gonzalez]

Hello, I agree with Roger, you should definitely try to increase the salt
concentration to get rid of non specific binding impurities. And if that
doesn't work, you can try purifying your protein under denaturing
conditions by adding one refolding step in the column.
Good luck,
Javier

On Wed, Dec 9, 2020 at 11:26 AM Roger Rowlett  wrote:

> Salt concentrations less than 100 mM can lead to nonspecific adsorption to
> the gel exclusion media, potentially leading to band broadening, and
> delayed elution.  Overloading gel exclusion columns (more than 2-4% Vt) can
> also lead to elution band artifacts. Check these issues first.
>
> Roger Rowlett
> Gordon & Dorothy Kline Professor, Emeritus
> Department of Chemistry
> Colgate University
>
> On Wed, Dec 9, 2020, 9:17 AM   wrote:
>
>> Dear All
>> There is a 43kd protein purified via Ni-chelating affinity
>> chromatography, anion exchange chromatography and gel filtration
>> chromatography in sequence. However the chromatogram obtained showed an
>> extremely asymmetric peak shape. The aggregation forms of proteins are
>> mainly in the range of monomers and dimers(Hepes and low concentration of
>> salt were used as buffers for gel filtration chromatography). 5% glycerinum
>> and 1mM Benzamidine hydrochloride had been added in order to maintain the
>> stability of the protein and prevent it from degrading. But well, all the
>> efforts seem to be useless. We wonder if there are any effective measures
>> can be taken to radically solve this problem. We would be much indebted for
>> the suggestions you offer.
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] Macports and Fink - failed building open source pymol on MacOS Catalina

[Javier Gonzalez]

Thank you so much Vijaykumar and Kevin, it worked smoothly!
Best wishes,
Javier

On Sun, Jun 14, 2020 at 5:04 PM Kevin Jin  wrote:

> My OS Version: Catalina 10.15.5;
>
> brew install brewsci/bio/pymol
>
> To verify, I just installed Pymol using brew 2 minutes ago. It works.
>
> On Sat, Jun 13, 2020 at 4:45 PM Javier Gonzalez  wrote:
>
>> Hello,
>> I'm attempting to build pymol on a laptop running MacOS Catalina 10.15.5,
>> Processor 2.8GHz Quad-Core Intel i7, Graphics Intel Iris Pro 1536 MB
>> I downloaded the latest version of Xcode 11.1
>>
>> I tried from Fink (fink install pymol-py27) and Macports (sudo port
>> install pymol), as indicated here:
>> https://pymolwiki.org/index.php/MAC_Install
>>
>> Both scripts run to download all dependencies but at the end fail with
>> different messages (see below). Any ideas? Is it doable or am I just
>> following outdated instructions?
>> Thanks in advance!
>> Javier
>>
>> Fink: fails at compiling term-readkey-pm5184-2.37-1
>>
>> -
>>
>> -
>> Failed: phase compiling: term-readkey-pm5184-2.37-1 failed
>>
>> Before reporting any errors, please run "fink selfupdate" and try again.
>> Also try using "fink configure" to set your maximum build jobs to 1 and
>> attempt to build the package again.
>> If you continue to have issues, please check to see if the FAQ on Fink's
>> website solves the problem.  If not, ask on one (not both, please) of
>> these mailing lists:
>>
>> The Fink Users List 
>> The Fink Beginners List ,
>>
>> with a carbon copy to the maintainer:
>>
>> Christian Schaffner 
>>
>> Note that this is preferable to emailing just the maintainer directly,
>> since most fink package maintainers do not have access to all possible
>> hardware and software configurations.
>>
>> Please try to include the complete error message in your report.  This
>> generally consists of a compiler line starting with e.g. "gcc" or "g++"
>> followed by the actual error output from the compiler.
>>
>> Also include the following system information:
>> Package manager version: 0.45.1
>> Distribution version: selfupdate-rsync Fri Jun 12 21:49:17 2020, 10.15,
>> x86_64
>> Trees: local/main stable/main
>> Xcode.app: 11.5
>> Xcode command-line tools: 11.5.0.0.1.1588476445
>> Max. Fink build jobs:  8
>>
>> -
>>
>> -
>> Macports: apparently there is an issue with py38-pyqt5
>> --->  Computing dependencies for pymol
>> The following dependencies will be installed:  py38-pyqt5
>> Continue? [Y/n]:
>> --->  Fetching archive for py38-pyqt5
>> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
>> https://packages.macports.org/py38-pyqt5
>> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
>> http://aus.us.packages.macports.org/macports/packages/py38-pyqt5
>> --->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
>> https://ywg.ca.packages.macports.org/mirror/macports/packages/py38-pyqt5
>> --->  Building py38-pyqt5
>> Error: Failed to build py38-pyqt5: command execution failed
>> Error: See
>> /opt/local/var/macports/logs/_opt_local_var_macports_sources_rsync.macports.org_macports_release_tarballs_ports_python_py-pyqt5/py38-pyqt5/main.log
>> for details.
>> Error: Follow https://guide.macports.org/#project.tickets to report a
>> bug.
>> Error: Processing of port pymol failed
>>
>> -
>>
>> -
>> --
>> Dr. Javier M. González
>> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
>> Universidad Nacional de Santiago del Estero (UNSE)
>> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
>> Santiago del Estero. Argentina
>> Tel: +54-(0385)-4238352
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/

[ccp4bb] Macports and Fink - failed building open source pymol on MacOS Catalina

[Javier Gonzalez]

Hello,
I'm attempting to build pymol on a laptop running MacOS Catalina 10.15.5,
Processor 2.8GHz Quad-Core Intel i7, Graphics Intel Iris Pro 1536 MB
I downloaded the latest version of Xcode 11.1

I tried from Fink (fink install pymol-py27) and Macports (sudo port install
pymol), as indicated here: https://pymolwiki.org/index.php/MAC_Install

Both scripts run to download all dependencies but at the end fail with
different messages (see below). Any ideas? Is it doable or am I just
following outdated instructions?
Thanks in advance!
Javier

Fink: fails at compiling term-readkey-pm5184-2.37-1
-
-
Failed: phase compiling: term-readkey-pm5184-2.37-1 failed

Before reporting any errors, please run "fink selfupdate" and try again.
Also try using "fink configure" to set your maximum build jobs to 1 and
attempt to build the package again.
If you continue to have issues, please check to see if the FAQ on Fink's
website solves the problem.  If not, ask on one (not both, please) of
these mailing lists:

The Fink Users List 
The Fink Beginners List ,

with a carbon copy to the maintainer:

Christian Schaffner 

Note that this is preferable to emailing just the maintainer directly,
since most fink package maintainers do not have access to all possible
hardware and software configurations.

Please try to include the complete error message in your report.  This
generally consists of a compiler line starting with e.g. "gcc" or "g++"
followed by the actual error output from the compiler.

Also include the following system information:
Package manager version: 0.45.1
Distribution version: selfupdate-rsync Fri Jun 12 21:49:17 2020, 10.15,
x86_64
Trees: local/main stable/main
Xcode.app: 11.5
Xcode command-line tools: 11.5.0.0.1.1588476445
Max. Fink build jobs:  8
-
-
Macports: apparently there is an issue with py38-pyqt5
--->  Computing dependencies for pymol
The following dependencies will be installed:  py38-pyqt5
Continue? [Y/n]:
--->  Fetching archive for py38-pyqt5
--->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
https://packages.macports.org/py38-pyqt5
--->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
http://aus.us.packages.macports.org/macports/packages/py38-pyqt5
--->  Attempting to fetch py38-pyqt5-5.14.2_0.darwin_19.x86_64.tbz2 from
https://ywg.ca.packages.macports.org/mirror/macports/packages/py38-pyqt5
--->  Building py38-pyqt5
Error: Failed to build py38-pyqt5: command execution failed
Error: See
/opt/local/var/macports/logs/_opt_local_var_macports_sources_rsync.macports.org_macports_release_tarballs_ports_python_py-pyqt5/py38-pyqt5/main.log
for details.
Error: Follow https://guide.macports.org/#project.tickets to report a bug.
Error: Processing of port pymol failed
-
-
-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] Vote for cryoEM

[Javier Gonzalez]

1. We need renowned scientists on Twitter because it is one of the best
ways to spread words worth reading, mostly at these times when so many
pseudo-scientists are influencers on social media (or worse, some even have
a show on Netflix).
2. I'm voting for Mass Spec right now
Cheers,
@_biojmg

On Tue, Mar 31, 2020 at 5:06 PM Guillaume Gaullier 
wrote:

> One remark about Jürgen’s comment (quoted below).
>
> It is great that well established scientists use Twitter, because by doing
> so they implicitly accept to be contacted publicly and to engage in public
> conversations (assuming their account is public, which is the default when
> you sign up on Twitter). Why is that great? Well, this tremendously lowers
> the barrier to engaging in discussions with established scientists,
> especially for early career researchers (like myself) who would not
> necessarily dare sending a direct email to more senior scientists. Posting
> to mailing lists such as the present one is no substitute to directly
> getting the personal attention of your field’s leading experts, like it can
> happen on Twitter.
>
> The main problem with Twitter is its business model that promotes the most
> outrageous (or "engaging", in Twitter lingo) content, click-bait and the
> like. But fundamentally, Twitter is only a communication tool just like
> this mailing list, being different by its focus on spontaneity instead of
> accurate archiving of conversations. I believe the spontaneity it brings is
> valuable, despite its toxic business model, which can be circumvented for
> the most part (by unfollowing accounts that post click-bait and by
> exercising a healthy amount of self-discipline, i.e. thinking twice before
> posting or reposting something potentially click-bait-y).
>
> Guillaume (@Guillawme)
>
>
> On 31 Mar 2020, at 21:00, Jurgen Bosch  wrote:
>
> I personally tweet, and I know a lot of well established scientists that
> tweet too.
>
> Don't pretend Twitter is only junk, there are a lot of serious scientist
> tweeting good and valuable information. True there are enough stupid people
> tweeting BS.
>
> Jürgen
>
> P.S. Follow me on Twitter @Bosch_Lab
>
> On Mar 31, 2020, at 2:01 PM, Gloria Borgstahl 
> wrote:
>
> I personally don't tweet.
>
>
> On Tue, Mar 31, 2020 at 12:21 PM Sweet, Robert <
> 27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Real Men (and possibly Women too) Don't Tweet.
>>
>> Bob
>>
>> 
>> From: CCP4 bulletin board  on behalf of James
>> Holton 
>> Sent: Tuesday, March 31, 2020 12:18 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Vote for cryoEM
>>
>> Allessandro,
>>
>> The link you provide directs to a website hosted at someting called "
>> twitter.com".  My spam filter flagged it as junk.
>>
>> -James Holton
>> MAD Scientist
>>
>> On 3/31/2020 8:41 AM, Alessandro Vannini wrote:
>> We are head to head with mass-spectrometry in the #JBCMethodsMadness
>> CHAMPIONSHIP. This can’t happen!
>>
>> Get out and VOTE! 15 min to go!
>>
>> https://twitter.com/jbiolchem/status/1244655631316987905?s=20<
>> https://urldefense.com/v3/__https://twitter.com/jbiolchem/status/1244655631316987905?s=20__;!!P4SdNyxKAPE!QXCVmZuWG8FEmwM9FbPP-f3LEbH6bMcORBgYwRGHCpxZF7cWEHW9OsXmS5uQ_Q$
>> >
>>
>> [cid:part2.4691618D.6AA0935A@lbl.gov]
>>
>> Sent from my iPhone
>>
>> 
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>
> --
>
> 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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-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352

Re: [ccp4bb] Protein cavity volume calculation

[Javier Gonzalez]

Hi Seema,
You can also have a look at https://www.caver.cz/ they´ve done a great job
with the CAVER program to calculate, analyze and visualize channels and
tunnels in macromolecules.
Best wishes,
Javier


On Sat, Jun 15, 2019 at 7:57 PM Seema H Irani 
wrote:

>
> I was wondering if anyone could recommend a good program or links to
> calculate the cavity volume of the protein active site.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] Re-using 96-well crystallization plates

[Javier Gonzalez]

Hello Nemanja,
I used to wash and reuse glass plates for neutron crystallography. Of
course glass is sturdier than polystyrene, but I can't think of any protein
stain that would resist a treatment with detergent, then a strong base (say
0.1M NaOH) and finally a strong acid (say 0.1M HCl)...
Regarding the drop shape problem that Janet mentioned, we applied Sigmacote
to the dry clean plates, a siliconizing agent sold by Sigma Aldrich, which
turns the surface non-adherent and chemically inert. From the website:
*Sigmacote® is a solution of a chlorinated organopolysiloxane in heptane
that readily forms a covalent, microscopically thin film on glass. The film
repels water, retards the clotting of blood or plasma, and prevents surface
adsorption of many basic proteins.*
However, I don't know whether polystyrene would resist the heptane solvent,
but the applied coat is very thin and should evaporate quickly if let dry
in a hood with the fan on.
I hope this helps and please let me know if it works!!
Best wishes,
Javier


On Wed, Apr 10, 2019 at 8:11 PM Newman, Janet (Manufacturing, Parkville)
 wrote:

> Hi Nemanja,
>
>
>
> I have tried doing this, and it has never really worked for me, even with
> careful rinsing with MilliQ water after washing, I could never get
> well-shaped drops on a recycled plate. They are also a real pain to wash
> out, and it’s hard to get the last traces of protein out of the subwells
> without scratching the subwells. (I was also doing this with the
> polystyrene SD-2 plates from SwissSci)
>
>
>
> Janet
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Nemanja
> Vuksanovic
> *Sent:* Thursday, 11 April 2019 4:42 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Re-using 96-well crystallization plates
>
>
>
> Dear All,
>
>
>
> I'd like to ask if anyone has experience cleaning old 96 well
> crystallization plates? I have a large number of old plates (Swissci) with
> mostly INDEX and PEG Ion screens and I thought of re-using them instead of
> throwing them away, but I'm not sure if this would be viable.
>
>
>
> Best regards,
>
>
>
> Nemanja Vuksanovic
>
>
> --
>
> Graduate Student
>
> Department of Chemistry and Biochemistry
>
> University of Wisconsin-Milwaukee
>
>
>
>
>
>
> --
>
> 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
>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  LinkedIn




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Re: [ccp4bb] Off topic question

[Javier Gonzalez]

Hi Reza, happy new year!
The choice would depend on your alignment (aminoacid or nucleotides? are
the sequences closely or distantly related? is it a large alignment? are
there many gaps?)... Anyway, I think the safest, unbiased way to determine
a group of outliers might be to compute a phylogenetic tree and look for an
outgroup. But if the set of sequences is too large you might want (first)
to use a clustering algorithm, such as CD-HIT (
http://weizhongli-lab.org/cd-hit/).
HTH,
Javier

On Thu, Jan 3, 2019 at 6:08 PM Ethan A Merritt 
wrote:

> On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote:
> > ?Hi,
> >
> >
> > Happy new year to all!  A bit of an off topic question.  Does anyone
> know of a method/program to extract the most distinct "n" (n>2) sequences
> from a sequence alignment?  Thanks.
>
> If these putative "most distinct" sequences are hypothesized to belong
> together,  then i suggest K-means clustering.  If they are hypothesized
> to be unrelated individual outliers then I think you would just take the
> worst scores using whatever metric you used to create original alignment.
>
> Ethan
>
> >
> >
> > Best wishes,
> > Reza
> >
> >
> > Reza Khayat, PhD
> > Assistant Professor
> > City College of New York
> > Department of Chemistry
> > New York, NY 10031
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> MS 357742,   University of Washington, Seattle 98195-7742
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  LinkedIn




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Re: [ccp4bb] nonenzymatic removal of His tag?

[Javier Gonzalez]

Another option would be to use a protease immobilized in some resin (for
instance GST-tagged TEV protease bound to GSH-Agarose resin, in column or
in batch), so that no protease would be free to co-purify with your protein
or peptide.
HTH,
Javier

On Thu, Sep 20, 2018 at 5:35 PM, Patrick Loll  wrote:

> 1) Use a different protease, e.g. SUMO protease, which is sufficiently
> specific that it’s unlikely to cause any problems even if a little bit is
> carried along. See, for example (shameless plug #1): DOI:
> 10.1016/j.pep.2006.12.006
>
> 2) Use intein cleavage. NEB sells a vector that lets you fuse an intein &
> a CBD at the C-term end of your POI; we’ve made a similar vector with a
> His-tag (shameless plug #2): DOI: 10.1021/ja208755j
>
> 3) Just assure your colleague that you guys are good protein chemists, so
> don’t worry, be happy.
>
> There are chemical cleavage methods (e.g. cyanogen bromide), but they
> aren’t particularly gentle, so if your 110-residue peptide is meant to fold
> into a native structure, I’d approach them with caution (on the other hand,
> if the peptide ISN’T meant to be folded, then just purify via
> reverse-phase, and you’ll for sure get rid of protease contaminants).
>
> Pat
>
>
> > On 20 Sep 2018, at 4:17 PM, Gloria Borgstahl 
> wrote:
> >
> > Hello, friends in crystallography,
> > A colleague just asked me this question.  He is worried about trace
> > protease interfering with the receptors he is studying in cell-based
> > experiments using a 110 amino acid protein we made for him.  He has
> > been unable to make the peptide synthetically.  The company is having
> > trouble getting that to happen.  Any ideas?  Happy Thursday, G
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> 
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  LinkedIn




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Re: [ccp4bb] Zn+2 proteases

[Javier Gonzalez]

Hello Jiri,
I wouldn't choose neither of them, but Co(II). NMR spectroscopists have
been using Co(II) to probe Zn(II) sites in proteins for ages... Check out
Bertini (*DOI: *10.1021/ar00092a002
), or Vallee (*DOI: *
10.1021/bi00476a001 ).
There are many examples of Zn(II) enzymes retaining their activity as
Co(II) surrogates, for instance metallo-beta-lactamases (*DOI: *
10.1021/bi100894r ).
Best wishes,
Javier

On Sun, Jul 29, 2018 at 6:13 PM, chemocev marker 
wrote:

> Dear All
>
> I have a questions about the Zn+2 proteases (Thermolysin based Zn+2 site)
>
> As Zinc is trouble metal when it comes to test the activity.
>
> Which is better substitute for it.
>
> Is MgCl2 or MncCl2
>
> Why one is preferred over the other.
>
> best
>
> Jiri
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  LinkedIn




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Re: [ccp4bb] capillary counterdiffusion agarose plug

[Javier Gonzalez]

Hello Georg,

You can melt the agarose in a glass container using a microwave oven. Then
connect your capillary tube to small syringe, using a plastic tubing or
adapter. Use some parafilm if the tubing diameters do not fit exactly,
there should be no leaks. Then slowly pull the plunger to take ~100ul of
agarose and keep pulling outside the liquid, until the plug is more or less
in the center of the tube, leaving room for the precipitant and protein on
each side.

But most importantly, bear in mind that the system in the paper you cite
works because the mother liquor components can diffuse readily through the
agarose plug (MPD, NaAc, AmSO4, CaCl2). This might not be the case if your
precipitant has high molecular weight PEG or the like, and your crystals
need lower temperature to grow.

Good luck,
Javier


On Mon, Jan 5, 2015 at 11:25 AM, Georg Mlynek georg.mly...@univie.ac.at
wrote:

 Dear Colleagues,

 After reading a few papers about growing  suitable crystals for neutron
 diffraction. I will do capillary counterdiffusion with an agarose plug
 between mother liquor and protein solution like described in
 http://www.ncbi.nlm.nih.gov/pubmed/23192028.

 However I looked quite some time now, to find how to put the 100 ul 1%
 low-melting agarose plug in the middle of the capillary, but was not
 successful.  Does one use a gel loading tip or is there a better way to do
 this?

 Thanks in advance for any tips and tricks,

 best regards, Georg.




-- 
Javier M. Gonzalez, PhD.
Protein Crystallography Station
Bioscience Division
Bioenergy and Biome Sciences Group (B-11)
Los Alamos National Laboratory
TA-3, Building 4200, Room 202B
Mailstop T007
Los Alamos, NM 87545
Phone: +1 (505) 667-9376
LinkedIn http://www.linkedin.com/pub/javier-gonz%C3%A1lez/22/7b/83a Email
bio...@gmail.com

Re: [ccp4bb] software/web server to determine ligand volume

[Javier Gonzalez]

You can calculate volumes and much more, here:

http://www.molinspiration.com/cgi-bin/properties

Javier


On Wed, Aug 13, 2014 at 12:06 AM, sreetama das somon_...@yahoo.co.in
wrote:

 Dear all,
 Is there any software or web server available to calculate the volume of a
 ligand if the ligand coordinates are provided?
 Google seems to come up only with options to calculate protein cavity
 volume.

 Thanks in advance,
 Sreetama Das,
 phd student,
 Physics, IISc




-- 
Javier M. Gonzalez, PhD.
Protein Crystallography Station
Bioscience Division
Bioenergy and Biome Sciences Group (B-11)
Los Alamos National Laboratory
TA-3, Building 4200, Room 202B
Mailstop T007
Los Alamos, NM 87545
Phone: +1 (505) 667-9376
LinkedIn http://www.linkedin.com/pub/javier-gonz%C3%A1lez/22/7b/83a Email
bio...@gmail.com

Re: [ccp4bb] Google Gets it Right

[Javier Gonzalez]

Interestingly, that molecule has the opposite configuration. The actual
modelhttp://upload.wikimedia.org/wikipedia/commons/6/6e/Molecular_model_of_Penicillin_by_Dorothy_Hodgkin_%289663803982%29.jpghas
a posting saying (wrong absolute configuration).
I wonder what's the story behind it...




On Mon, May 12, 2014 at 2:28 PM, Gregg Crichlow gregg.crich...@gmail.comwrote:

 Actually, it was noticing penG that made me mouse over it myself. After
 spending many years completing a thesis on beta-lactamases, I was very
 surprised - and excited - to see that on something as main-stream as
 Google. But then when I paid attention more closely, I saw that they even
 have a good representation of the electron density in three orthogonal
 planes surrounding the molecule!  Dr. James Knox (my thesis advisor) just
 informed me that the density maps are on exhibit at the British Museum of
 Science.
   I have heard so much about Dorothy Hodgkin from my thesis advisor,
 that I became very familiar with her name and legacy, although I never had
 met her.

 Gregg


 On Mon, May 12, 2014 at 9:39 AM, Robert Sweet sw...@bnl.gov wrote:

 Check out google.com.  They're announcing what would have been Dorothy
 Hodgkin's 104th b-day.  I saw the molecule, and said to myself, My
 goodness, that's penicillin G, and the mouseover announced the big day.

 She was a great scientist and a friend to thousands of us.

 Bob

 =
 Robert M. Sweet E-Dress: sw...@bnl.gov
 Group Leader, PXRR: Macromolecular   ^ (that's L
   Crystallography Research Resource at NSLSnot 1)
   http://px.nsls.bnl.gov/
 Photon Sciences and Biosciences Dept
 Office and mail, Bldg 745, a.k.a. LOB-5
 Brookhaven Nat'l Lab.   Phones:
 Upton, NY  11973631 344 3401  (Office)
 U.S.A.  631 344 2741  (Facsimile)
 =





-- 
Javier M. Gonzalez, PhD.
Protein Crystallography Station
Bioscience Division
Bioenergy and Biome Sciences Group (B-11)
Los Alamos National Laboratory
TA-3, Building 4200, Room 202B
Mailstop T007
Los Alamos, NM 87545
Phone: +1 (505) 667-9376
LinkedIn http://www.linkedin.com/pub/javier-gonz%C3%A1lez/22/7b/83a
Emailbio...@gmail.com

[ccp4bb] Usage of gels in protein crystallography

[Javier Gonzalez]

Dear CCP4BBers,

I would very much appreciate any information or resources regarding usage
of gels in order to achieve supersaturation/crystallization through liquid
diffusion. It appears to me that, although this crystallization method is
usually claimed as a powerful technique, it is very limited in practice.
For instance, PEG solutions display little to no diffusion through gel
matrices, and some gels may undergo significant changes in volume (and
thereby their average pore size) depending on the medium ionic strength and
salt composition, leading to significant loss of protein.

Thanks in advance,
Javier

-- 
Javier M. Gonzalez, PhD.
Protein Crystallography Station
Bioscience Division
Bioenergy and Biome Sciences Group (B-11)
Los Alamos National Laboratory
TA-3, Building 4200, Room 202B
Mailstop T007
Los Alamos, NM 87545
Phone: +1 (505) 667-9376
LinkedIn http://www.linkedin.com/pub/javier-gonz%C3%A1lez/22/7b/83a
Emailbio...@gmail.com

[ccp4bb] Are there.cif dictionaries for deuterated aminoacids for Coot?

[Javier Gonzalez]

Dear all,
I'm refining a neutron structure and I would like to know whether there are
available geometry dictionaries for deuterated aminoacids, in order to
input them into Coot. At least the version I have (Coot 0.7, revision 4459)
is unable to handle aminoacids whose exchangeable H atoms are replaced by D.
I would rather avoid the hassle of having to edit manually the .cif files
for all aminoacids.

Thanks in advance,
Javier

-- 
Javier M. Gonzalez
 Protein Crystallography Station
Bioscience Division
Los Alamos National Laboratory
TA-3, Building 4200, Room 202
Mailstop T001
work: (505) 667-9376 (temporary)

Re: [ccp4bb] vitrification vs freezing

[Javier Gonzalez]

Hi Sebastiano,

I think the term vitrified crystal could be understood as a very nice
oxymoron (http://www.oxymoronlist.com/), but it is essentially
self-contradictory and not technically correct.

As Ethan said, vitrify means turn into glass. Now, a glass state is a
disordered solid state by definition, then it can't be a crystal. A
vitrified crystal would be a crystal which has lost all three-dimensional
ordering, pretty much like the material one gets when using the wrong
cryo-protectant.

What one usually does is to soak the crystal in a cryo-protectant and
then flash-freeze the resulting material, hoping that the crystal structure
will be preserved, while the rest remains disordered in a solid state
(vitrified), so that it won't produce a diffraction pattern by itself, and
will hold the crystal in a fixed position (very convenient for data
collection).

Moreover, I would say that clarifying a material is vitrified when
subjected to liquid N2 temperatures would be required only if you were
working with some liquid solvent which might remain in the liquid phase at
that temperature, instead of the usual solid disordered state, but this is
never the case with protein crystals.

So, I vote for frozen crystal.-

Javier


PS: that comment by James Stroud I forgot to mention that if any
dictionary is an authority on the very cold, it would be the Penguin
dictionary., is hilarious, we need a Like button in the CCP4bb list!

--
Javier M. Gonzalez
Protein Crystallography Station
Bioscience Division
Los Alamos National Laboratory
TA-43, Building 1, Room 172-G
Mailstop M888
Phone: (505) 667-9376


On Thu, Nov 15, 2012 at 2:24 PM, Craig Bingman cbing...@biochem.wisc.eduwrote:

  cryopreserved

 It says that the crystals were transferred to cryogenic temperatures in an
 attempt to increase their lifetime in the beam, and avoids all of the other
 problems with all of the other language described.

 I was really trying to stay out of this, because I understand what
 everyone means with all of their other word choices.

 On Nov 15, 2012, at 2:07 PM, James Stroud wrote:

  Isn't cryo-cooled redundant?
 
  James
 
  On Nov 15, 2012, at 11:34 AM, Phil Jeffrey wrote:
 
  Perhaps it's an artisan organic locavore fruit cake.
 
  Either way, your *crystal* is not vitrified.  The solvent in your
 crystal might be glassy but your protein better still hold crystalline
 order (cf. ice) or you've wasted your time.
 
  Ergo, cryo-cooled is the description to use.
 
  Phil Jeffrey
  Princeton
 
  On 11/15/12 1:14 PM, Nukri Sanishvili wrote:
  s: An alternative way to avoid the argument and discussion all together
  is to use cryo-cooled.
  Tim: You go to a restaurant, spend all that time and money and order a
  fruitcake?
  Cheers,
  N.
 

[ccp4bb] Job Posting - Laboratory Specialist in NMR, São Paulo University, Brazil

[Javier Gonzalez]

This is a forwarded message. For inquires please contact chsfa...@iq.usp.bror
robe...@iq.usp.br
___

The Central Analítica at the Instituto de Química, Universidade de São
Paulo has an opening for a Laboratory Specialist in NMR. The position is
associated with the acquisition of an 800 MHz magnet destined to study
biological macromolecules.

Applications will be accepted up to 31 of January, 2012. The full-time
position (40 hrs/week) comes with a salary of R$ 5691,08, plus benefits.

More information regarding the position can be found below, in the
following link http://www2.iq.usp.br/rh/ - link: VAGAS/Editais and by
writing to chsfa...@iq.usp.br or robe...@iq.usp.br.

We ask for your help in publicizing this opportunity among the
undergraduate and graduate students and post-doctoral fellows in your
scientific community.

Best regards,

Chuck Farah and Roberto Salinas
Instituto de Química, USP
___

A Central Analítica do Instituto de Química da USP tem uma vaga para
Especialista de Laboratório em RMN. A vaga está associada à aquisição de um
magneto de 800 MHz para estudo de macromoléculas biológicas.

A contratação será sob o Regime da CLT em jornada de trabalho de 40 horas
semanais. O salário para o mês de outubro/2011 é de R$ 5.691,08, mais
benefícios.

Mais informações podem ser obtidas no site http://www2.iq.usp.br/rh/ -
link: VAGAS/Editais e escrevendo para chsfa...@iq.usp.br ou
robe...@iq.usp.br.

Pedimos sua ajuda na divulgação desta oportunidade entre alunos de
graduação, pós-graduação e pós-doutores da sua comunidade científica.
___


Vaga Para Contratação de Técnico de Nível Superior Associada à Operação de
Instrumento de RMN de 800 MHz no Instituto de Química da Universidade de
São Paulo.

O Instituto de Química da Universidade de São Paulo torna pública a
abertura de concurso público para preenchimento de 01 vaga na carreira do
Grupo Superior S1 A para a função de Especialista em Laboratório, vinculada
ao Programa de Concessão de Técnico de Nível Superior para Grupos de
Excelência - PROCONTES da Pró-Reitoria de Pesquisa da Universidade de São
Paulo, que visa atender projetos especiais de pesquisa por ela selecionados.

A vaga destina-se ao projeto de Aquisição de um Instrumento de Ressonância
Magnética Nuclear de 800 MHz com Sonda Resfriada para a Central Analítica
do Instituto de Química da USP.

A vaga é destinada a jovens cientistas com Curso de Graduação completa em
Química, Farmácia, Física, Engenharia, Biomedicina ou Biologia com carga
horária mínina fixada pelo MEC. Candidatos devem ser fluentes em inglês e
possuir experiência na operação de instrumentação analítica, em especial
equipamentos de Ressonância Magnética Nuclear.

A contratação será sob o Regime da CLT em jornada de trabalho de 40 horas
semanais. O salário para o mês de outubro/2011 é de R$ 5.691,08, mais
benefícios.

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link: VAGAS/Editais e escrevendo para chsfa...@iq.usp.br ou
robe...@iq.usp.br.

Re: [ccp4bb] how to remove part of data with bad signal to noise ratio

[Javier Gonzalez]

I would also make sure that the spacegroup is correct. If you have instead
P222, P2212, etc, you might find the solution at low resolution but the
problem would become evident during advanced refinement steps, such as a
stuck high Rfree or a noisy difference map.
Good luck,
Javier.

On Sat, May 21, 2011 at 4:44 PM, Seema Mittal seema.mit...@umassmed.eduwrote:

 Thank you all for your thoughts and suggestions in response to my query.

 The rotamers, peptide omega angles, mol-probity data all are perfectly
 fine. There are 3 outliers in Ramachandran plot ( may be the ones causing
 the problem).
 The protein has two engineered cysteines involved in disulfide bond
 formation which restrict the conformation around the disulfide bond. One of
 these cys residues is followed by pro which seems to be constrained a bit.
 Nevertheless, the overall geometry is good.

 There certainly are a couple of big positive density peaks which i haven't
 been able to account for yet. The usual suspects from my crystallization
 conditions do not seem to fit in those blobs.

 I shall go back to 2.7A resolution and continue to try to resolve these
 positive peaks. Thanks for your suggestions. Any other thoughts on the
 matter would be greatly appreciated.

 Thanks much,
 Best,
 Seema



 On May 20, 2011, at 10:41 PM, Ethan Merritt wrote:

  On Friday, 20 May 2011, you wrote:

 Hi Ethan,

 You are absolutely right. As a matter of fact, I had initially
 processed the data to 2.7A and it looked pretty decent with R symm
 less than 10%. The maps looked good too.

 The problem arose during second round of refinement. The Rfree got
 stuck at around 29-30 while the Rfactor kept decreasing to about
 20-21.



 That can simply mean there are many little things wrong with the
 structure - rotamers, phi/psi, perhaps even a register shift.
 I suggest that you use the 2.7A data, so that there is more local
 information to drive the refinement, and take seriously any
 hints that Molprobity provides about bad local geometry.

 Another thought - are there any large positive peaks in the
 difference density?  Perhaps you are missing a solvent ion or
 something of that sort.  Even one missing sulfate could make
 a noticeable difference in Rfree.

regards,

Ethan



  The bond length and angle values are fine too.

 I cut down the resolution to 3A hoping to improve the data quality by
 removing some noise. But, it did not work. i also tried to put
 restrains on the backbone B factors with limited success.

 Any thoughts on how i can resolve this Rfree issue?

 Thanks much,
 Seema



 On May 20, 2011, at 5:38 PM, Ethan Merritt wrote:

  On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote:

 Hi All,

 I am currently working on a 3A resolution dataset. The scaled file
 shows the following statistics (scroll down to the end of this
 email). It is P212121 space group with R merge of 8.8%.


 Your data statistics look fine.  In fact, it looks to me that your
 crystal is
 probably yielding good data to considerably better resolution than 3A.
 Why did you choose to cut it there?

  My question is : Is there a way to selectively use only the data
 with  I/Sigma value of 2 and more for refinement?


 That is a bad idea.  By removing data you are throwing away
 information.
 Noisy data is still better than no data.

good luck with your [probably better than 3A] structure,

Ethan


  And how do i achieve this using refmac? I am aware that this would
 come at the cost of compromising data completeness. Any
 suggestions/help would be greatly appreciated.


 Thanks much,
 Seema Mittal
 Department of Biochemistry  Molecular Pharmacology
 970L Lazare Research Building
 University of Massachusetts Medical School
 364 Plantation Street
 Worcester, MA 01605





 Shell I/Sigma in resolution shells:
  LowerUpper  % of of reflections with I / Sigma less than
  limit limit 0 1   2  3  5
 10 2020 total
  50.00 6.46   2.0   3.8   5.3   6.27.6   12.5   34.3
 65.0   99.3
   6.46  5.13   0.7   2.2   3.9   5.38.2   15.7   36.6
 63.4  100.0
   5.13  4.48   1.3   2.8   4.0   5.89.3   13.8   27.3
 72.7  100.0
   4.48  4.07   0.7   1.7   4.0   5.47.9   13.9   35.4
 64.1   99.5
   4.07  3.78   1.8   3.6   5.1   6.9   11.8   20.8  49.6
 47.3   96.9
   3.78  3.56   1.5   3.8   6.7   8.7   13.3   26.7  65.4
 30.8   96.2
   3.56  3.38   0.8   3.2   7.1   8.9   12.9   31.1  76.6
 20.0   96.6
   3.38  3.23   2.0   4.8   8.1  14.8  23.4   44.8  84.7
 12.7   97.5
   3.23  3.11   4.1   9.2  13.8  18.4  29.6  51.0  86.0
 11.0   96.9
   3.11  3.00   2.4   8.6  13.9  18.8  30.6  53.9  92.4
 4.596.9
  All hkl1.7  4.3   7.19.8   15.3   28.0
 58.1   39.9   98.0


  Shell Lower Upper Average  Average Norm. Linear Square
  limitAngstrom   I   error   stat. Chi**2  

Re: [ccp4bb] LIGPLOT or similar

[Javier Gonzalez]

Mark, you might want to try PDBsum (http://www.ebi.ac.uk/pdbsum/), it will
generate a LIGPLOT output for a given PDB code entered, which can be
downloaded as high resolution .pdf or .ps
Best,
Javier

On Wed, Aug 25, 2010 at 2:49 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 Hello Mark,

 if you would describe the error message you get from the UNIX version of
 LIGPLOT, someone on the list might be able to help you while you have to
 wait
 for and apple installer version or the (commercial) alternatives.

 Looking at the pictures about ligplot I found on the web and considering
 what I
 learned about pymol recently you might also be able to produce pictures
 with
 similar information content with pymol, although the pictures would be
 neatly
 rendered.

 Best wishes, Tim

 On Wed, Aug 25, 2010 at 07:51:08PM +0200, Mark J van Raaij wrote:
  Dear All,
 
  Having just installed LIGPLOT under Windows, I find it rather convoluted
 to run. It has to be run via de command line window, and I try to avoid
 Windows as much as I can anyway.
  I also tried to install the Unix version on MacOSX, but was not able to
 get it running properly, probably at least partly due to my relative lack of
 informatics skills...
 
  Is there an alternative program that does the same (pref. with MacOSX
 version)?
 
  For the LIGPLOT developers, ideal would be a MacOSX installer (dmg) - I
 think it would lead to more use of your program.
 
  Greetings,
 
  Mark van Raaij

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

 GPG Key ID = A46BEE1A


 -BEGIN PGP SIGNATURE-
 Version: GnuPG v1.4.9 (GNU/Linux)

 iD8DBQFMdWXHUxlJ7aRr7hoRAhulAKC332MIxmaHPVu8lnhL6dL0GyYE0wCffnJ2
 6juBxgbS93ld7NV6ALKcDm0=
 =GHc4
 -END PGP SIGNATURE-




-- 
Javier M. Gonzalez, PhD.
University of Maryland Baltimore
Department of Pharmaceutical Sciences
X-Ray Crystallography Shared Service (UMXSS)
20 Penn St., HSFII, Rm 514
Phone/Fax: 410-7061124/410-7060886
21201 Baltimore, MD
http://www2.pharmacy.umaryland.edu/psc/xray/

Re: [ccp4bb] attachments

[Javier Gonzalez]

Dear all,
   I've just created a group on Facebook named CCP4 Stuff. This would be a
good place to upload as much random stuff as you want (images, videos,
links, comments, etc) that nobody will care about after a couple of days.
All you need is to join (this is not an issue, considering that most
uploaders are grad students and postdocs asking for advice, who probably
have a Facebook account) and then paste the link in your CCP4bb email text,
like this

http://www.facebook.com/photo.php?pid=59394l=6139f5a298id=11221895783

Since the group is open, a Facebook account is not needed to have access
to the uploads once they are created.

Best,

Javier

Re: [ccp4bb] How could I Extract Iron from Protein?

[Javier Gonzalez]

Since you have only Asp and His ligands coordinating the Fe ion, dialysis
against, say, 0.1 M Citrate at pH 5 will do the trick. Citrate will chelate
the Fe(III) (if the protein has Fe(II) it will be oxidized during dialysis
due to air oxygen) avoiding precipitation of Fe(OH)3 and the acid will
protonate the protein ligands. If the affinity is really high, adding also
some guanidinium chloride (0.5-1M) in the first dialysis step to loosen the
protein a little bit will also help.

Best,

Javier M. Gonzalez, PhD.
University of Maryland Baltimore
Department of Pharmaceutical Sciences
X-Ray Crystallography Shared Service (UMXSS)
20 Penn St., HSFII, Rm 514
Phone/Fax: 410-7061124/410-7060886
21201 Baltimore, MD
http://www2.pharmacy.umaryland.edu/psc/xray/



On Tue, May 11, 2010 at 12:26 PM, Roger Rowlett rrowl...@colgate.eduwrote:

  I would try dialyzing against a solution with 1,10-phenanthroline, 
 1,10-phenanthroline-2-carboxylate,
 or pyridine-2,6-dicarboxylate. Removal of metals from proteins is often not
 just dissociative, but requires the associative interaction of a chelating
 agent. Which one works is often empirical.

 Cheers.


 On 5/11/2010 11:11 AM, Wenguang LIANG wrote:

Dear all,

 I have a protein which binds iron with two D and two H as active site. I
 have tried to extract the iron by dialysis. First, 20mM tris, pH7.5, 150mM
 NaCl, 20EDTA, 10mM Na2S2O3, O/N. Then, followed by dialysis with 20mM tris,
 pH7.5, 150mM NaCl, 1EDTA O/N to remove the EDTA and Na2S2O3. However, after
 dialysis, the density of the Iron is still in the protein.

 Could anybody please give me some suggestion on how to extract Iron out of
 the protein? Or I can just use Chelex-100 to extract it instead of Iron.

 Thank you very much for help,

 Best wishes,

 Vinson Liang



 --
  --
 Roger S. Rowlett
 Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346

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