[ccp4bb] CCP4 job list disappeared

[KL Ho]

Hi All,

This is a bit off topic I guess. The system I am using is a dual-boot
system with Windows 8 and Ubuntu 12.1 installed. I am running CCP4 in
Ubuntu in a shared folder (File system: NTFS) with windows. Before this
morning, everything was fine except that the system could not read
(invisible) the text files that were created in windows. I tried to run
CCP4 this morning, I found that the job list disappeared although the
CCP4_database folder and all the files are still there. Another strange
problem is some of the folders created by Phenix are not visible in that
folder after rebooting into windows 8. Is this something to do with
permission? Any comments would be appreciated. Thanks in advance.

Kok Lian

Re: [ccp4bb] CCP4 job list disappeared

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Kok Lian,

can you run a filesystem check from the windows side? I don't know the
current status of the NTFS driver in Linux, but it used to be quite
broken (unsurprisingly, given NTFS ;-) and I would not write data on
an NTFS partition using Linux still. Maybe Linux scrambled up
something which can be recovered by a filesystem check.

Regards,
Tim

On 03/14/2014 09:23 AM, KL Ho wrote:
> Hi All,
> 
> This is a bit off topic I guess. The system I am using is a
> dual-boot system with Windows 8 and Ubuntu 12.1 installed. I am
> running CCP4 in Ubuntu in a shared folder (File system: NTFS) with
> windows. Before this morning, everything was fine except that the
> system could not read (invisible) the text files that were created
> in windows. I tried to run CCP4 this morning, I found that the job
> list disappeared although the CCP4_database folder and all the
> files are still there. Another strange problem is some of the
> folders created by Phenix are not visible in that folder after
> rebooting into windows 8. Is this something to do with permission?
> Any comments would be appreciated. Thanks in advance.
> 
> Kok Lian
> 

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

GPG Key ID = A46BEE1A

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Comment: Using GnuPG with Icedove - http://www.enigmail.net/

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[ccp4bb] pairwise CCano

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear all,

I am looking for a tool that prints (and preferably plots, e.g. as
postscript) the pairwise anomalous CC vs. resolution for several input
HKL-files.
xprep does this, but it is interactive and requires a fair bit of
typing. Since I have a large number of HKL-files from XDS, I would like
to script that and then flip through the pages of the postscript-plots.

I looked into pointless but could not find even a table.

Since there recently were some publications one the use of many files
for phasing, I though such a tool should exist!?

Best,
Tim

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFTIszcUxlJ7aRr7hoRApYyAKDt36pF11DAbkfcXVq+uLjqvm91HgCfSZ+V
BVpiCx4q9DZZCqDLenHX374=
=itCO
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Re: [ccp4bb] pairwise CCano

[LEGRAND Pierre]

Hello Tim :-)

You can try to do this with sftools. It is also an interactive type of program 
input but you can easily calculate correlations. After converting xds files to 
mtz, you can try some thing like this : 

sftools << eof > sftools_1.log
READ $mtz1
READ $mtz2
SELECT RESOL > 2.8
CORREL COL  5 10 SHELLS 10
eof

For the ploting, I'll use gnuplot after parsing the sftools_1.log file.
Good luck with this,
Cheers,
Pierre


De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Tim Gruene 
[t...@shelx.uni-ac.gwdg.de]
Envoyé : vendredi 14 mars 2014 10:33
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] pairwise CCano

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear all,

I am looking for a tool that prints (and preferably plots, e.g. as
postscript) the pairwise anomalous CC vs. resolution for several input
HKL-files.
xprep does this, but it is interactive and requires a fair bit of
typing. Since I have a large number of HKL-files from XDS, I would like
to script that and then flip through the pages of the postscript-plots.

I looked into pointless but could not find even a table.

Since there recently were some publications one the use of many files
for phasing, I though such a tool should exist!?

Best,
Tim

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFTIszcUxlJ7aRr7hoRApYyAKDt36pF11DAbkfcXVq+uLjqvm91HgCfSZ+V
BVpiCx4q9DZZCqDLenHX374=
=itCO
-END PGP SIGNATURE-

Re: [ccp4bb] pairwise CCano

[Phil Evans]

If you assigns them to different datasets in Pointless, then Aimless will give 
you the cross-dataset correlations. By default it will scale them to together 
first, but you can skip that if you want

It might not scale well to a large number of files (OK up to about 10 I guess)

Phil

On 14 Mar 2014, at 09:33, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear all,
> 
> I am looking for a tool that prints (and preferably plots, e.g. as
> postscript) the pairwise anomalous CC vs. resolution for several input
> HKL-files.
> xprep does this, but it is interactive and requires a fair bit of
> typing. Since I have a large number of HKL-files from XDS, I would like
> to script that and then flip through the pages of the postscript-plots.
> 
> I looked into pointless but could not find even a table.
> 
> Since there recently were some publications one the use of many files
> for phasing, I though such a tool should exist!?
> 
> Best,
> Tim
> 
> - -- 
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Icedove - http://www.enigmail.net/
> 
> iD8DBQFTIszcUxlJ7aRr7hoRApYyAKDt36pF11DAbkfcXVq+uLjqvm91HgCfSZ+V
> BVpiCx4q9DZZCqDLenHX374=
> =itCO
> -END PGP SIGNATURE-

[ccp4bb] Fwd: [ibs.tous] Les Houches SUMMER SCHOOL LAST CALL

[Isabel Garcia-Saez]

Dear all,
Find enclosed the last call for the Summer School on Integrated Biology to be 
held next July in the French Alps.
Best regards,
Isabel

Début du message réexpédié :

> De : Eva Pebay-Peyroula 
> Objet : [ibs.tous] Les Houches SUMMER SCHOOL LAST CALL
> Date : 14 mars 2014 08:10:17 HNEC
> 
> Dear All
> please find below an announcement for the Summer school on Integrated 
> structural and cell biology that we organize this summer. The Topics 
> developed in the school are of high importance for the future of the field. 
> For young scientist, it will give you new insights that might be essential 
> for your future carrier. We still have a few fellowships available.
> If you are interested, please tell me ASAP.
> 
> Best regards
> Eva
> 
> 
> 
> This is an announcement for a summer school organizied this summer in one of 
> the most gorgeous French locations: the Chamonix Valley. It's intended for 
> PhD students, post-docs and researchers at early stages of their career. It 
> deals with integrated structural cell biology, so really going from the atom 
> to the whole organism. The two biological processes used as models will be 
> viral infection and flower development but they are just examples and this 
> school is intended for all types of biologists of physicists interested in 
> biology. Participants will learn how a biological question can be answered 
> using an integrated approach and learn more about the methods. It's only 
> 1500€ for 4 weeks and there will be fellowships to cut the cost down to 750€. 
> So, please advertise it around you: there are only 50 spots!
> 
> To know more: http://leshouches2014.eu/
> 
> Location: Ecole des Houches
> 
> Dates: 07/07/14 to 01/08/14
> 
> Contact: Eva Pebay-Peyroula eva.pebay-peyro...@ibs.fr
> 
>  -- 
> __
> 
> JULY 2014
> Please note the summer school in the Mont-Blanc valley on Integrated 
> structural cell Biology.
> To know more see http://leshouches2014.eu/
> __
> 
> Prof. Eva PEBAY-PEYROULA, Dir.
> INSTITUT DE BIOLOGIE STRUCTURALE
> UMR5075 CEA-CNRS-UniversitŽ Joseph Fourier
> 6, rue Jules Horowitz
> F-38000 Grenoble
> Tel: 33(0)4 57 42 85 34 
> Mobile: 33(0)6 09 03 87 79
> email: eva.pebay-peyro...@ibs.fr
> 
> 

[ccp4bb] ITC with unfolded proteins

[Anita P]

Hello everyone,

I have a query for the scientists working on protein-protein interaction.
It is known that some proteins exist in unfolded or molten globule state
and attain structure on interaction with other folded proteins.
Many a times, it is difficult to obtain the structure of these complexes.

Is it possible to quantitatively determine the thermodynamics of
interaction between an unfolded protein and a folded protein using ITC?
Later may be perform an alascan to determine the residues of the unfolded
partner involved in the interaction.

Please share your ideas

cheers
Anita

[ccp4bb] New Frontiers in Neutron Macromolecular Crystallography Workshop July 15-16, 2014

[Leighton Coates]

New Frontiers in Neutron Macromolecular Crystallography Workshop

Oak Ridge National Laboratory

Spallation Neutron Source

USA

July 15-16, 2014



This meeting will bring together scientists to discuss new
opportunities for research at the two advanced neutron user facilities
(SNS and HFIR) at the Department of Energy’s (DOE) Oak Ridge National
Laboratory (ORNL). Users now have access to some of the world’s most
intense neutron beamlines for studying the structure of biological
systems using neutron macromolecular crystallography (NMC).

The advent of the SNS and the construction of MaNDi provide a new and
exciting opportunity for NMC to become a routine and essential
structural tool for enzymology, structural biology and functional
genomics. In order to satisfy the needs of the structural biology
community, a dedicated, high-resolution time-of-flight single crystal
macromolecular neutron diffractometer (MaNDi) has been constructed at
the SNS and is now operational.


An important function of the meeting will be to introduce current and
future MNC users to the new beamlines at ORNL and provide lectures on
structure refinement using PHENIX and nCNS. New developments will be
described that greatly enhance structure refinement.



Scholarships for attendance are available from:

• Joint Institute for Neutron Sciences for those
attendees from EPSCOR states (Alabama, Alaska, Arkansas, Delaware,
Hawaii, Idaho, Iowa, Kansas, Kentucky, Louisiana, Maine, Mississippi,
Montana, Nebraska, Nevada, New Mexico, North Dakota, Oklahoma, Rhode
Island, South Carolina, South Dakota, Tennessee, Utah, Vermont, West
Virginia, Wyoming, the Commonwealth of Puerto Rico, and the Virgin
Islands). For details, please visit
http://jins.tennessee.edu/epscor/index.html for the application form
and contact Hope Moore at hmoo...@utk.edu.

• The ORAU Travel Grants Program provides up to $800
to facilitate travel by a faculty member from an ORAU Sponsoring or
Associate Institution or Branch Campus. Visits can be to collaborate
with researchers at ORNL, Y-12, ORAU laboratories or work sites, or
another ORAU institution. To apply, visit the ORAU Members
Universities page, find your school, and contact your member
councilor, who can submit a proposal for travel funding to the ORAU
University Partnerships Office through the Members Only section of
that site.



More information can be found at this link http://neutrons.ornl.gov/conf/nmc2014





Leighton Coates

Biology and Soft Matter Division

Oak Ridge National Laboratory



P.O. Box 2008, Bldg. 8600, MS-6475

Oak Ridge, TN  37831-6475

E-mail coat...@ornl.gov

Re: [ccp4bb] off-topic: protein losing FAD during purification

[Boaz Shaanan]

Hi Stefano,

On top of all that has been suggested you should also be aware of the effect of 
pH and buffer composition on the apo-holoenzyme equilibrium during purification 
and crystallization.

 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  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Benini Stefano 
(P) [stefano.ben...@unibz.it]
Sent: Friday, March 14, 2014 12:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: protein losing FAD during purification

Dear All (those dealing with wetlab stuff..),

While purifying a FAD containing protein we lose part of the FAD (on the gel 
filtration we clearly see two bands corresponding to holoprotein and free FAD).

We obtain crystals but diffracting to only about 4 A despite their beautiful 
look. Our hypothesis is that the crystals contain a population of molecules 
with and without FAD (?).

The questions are:

1) how to keep FAD bound to the protein during purification and crystallization?

2) how to completely remove FAD from the protein?

Thank you very much for any help provided!

Best regards

Stefano (part-time wetlab person)


Dr Stefano Benini, Ph.D.
Assistant Professor

First International workshop: "Molecular Basis of Fire Blight", Bolzano 
15.10.2014

Laboratory homepage:
http://pro.unibz.it/staff2/sbenini/B2Cl.htm

Personal homepage
http://pro.unibz.it/staff2/sbenini/

"I don't like anything that's fake and I hate pretenders!"

*
Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl)
Faculty of Science and Technology
Free University of Bolzano
Piazza Università, 5
39100 Bolzano, Italy
Office (room K2.14):  +39 0471 017128
Laboratory (room E.021): +39 0471 017910
Fax: +39 0471 017009

"ogni giorno in più è un giorno in meno."

Re: [ccp4bb] ITC with unfolded proteins

[Reza Khayat]

Hi,

I think the experiment is doable, but how would you decouple 
protein-protein interaction from folding of the unfolded 
protein due to protein interaction?

Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
>Date: Fri, 14 Mar 2014 18:07:48 +0530
>From: CCP4 bulletin board  (on behalf 
of Anita P )
>Subject: [ccp4bb] ITC with unfolded proteins  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hello everyone,
>   I have a query for the scientists working on
>   protein-protein interaction.
>   It is known that some proteins exist in unfolded or
>   molten globule state and attain structure on
>   interaction with other folded proteins.
>   Many a times, it is difficult to obtain the
>   structure of these complexes.
>   Is it possible to quantitatively determine the
>   thermodynamics of interaction between an unfolded
>   protein and a folded protein using ITC? Later may be
>   perform an alascan to determine the residues of the
>   unfolded partner involved in the interaction.
>   Please share your ideas
>   cheers**
>   Anita

Re: [ccp4bb] off-topic: protein losing FAD during purification

[R. M. Garavito]

Stefano,

Before you address the problem, you need to ask yourself a couple of things. 

You say that "on the gel filtration we clearly see two bands corresponding to 
holoprotein and free FAD."  That is not too odd, but have you ask the question 
is all the protein good protein.  

Is this an enzyme? If so, assay samples before and after the gel filtration 
column without adding extra FAD and compare to assays with a slight excess FAD. 
 If the specific activities are different (slight excess FAD >  before gel 
filtration & no added FAD >  after the gel filtration & no added FAD), I would 
follow the good advice of the others.  You have just removed exchangeable FAD 
and are crystallizing a mixed system.

 Is this a recombinant protein partially purified using a His-tag or such?

If the specific activities are the same, I would suspect that not all the 
protein is in good shape.  Try ion exchange chromatography to see if two 
different protein variants can be separated.  With luck that can be (1) active 
holoprotein and "poorly-folded" protein (always a problem with His-tag purified 
recombinant protein) or (2) active holoprotein and apo-protein.

If it is not an enzyme, or it is not an easy assay, take the spectrum of the 
gel filtration-purified holoprotein and see if it differs from free FAD, then 
add FAD to a near stoichiometric level to see if you can saturate without a 
spectral change (e.g., a blue-shift of the absorption peak). That could tell 
you if it is an active holoprotein and apo-protein problem or active 
holoprotein and "poorly-folded" protein problem.  Sometimes  "poorly-folded" 
protein weakly binds the cofactors, but not in the native way.

If you are lucky, supplementing with FAD, either before, during, or after 
purification will work fine.  In some cases, I have found that it is better to 
purify the apo-protein, then reconstitute the holo-protein, if the former is 
stable enough.  However, some heme, NAD(P)(H), and FAD binding protein allow 
removal of the cofactors but not its reconstitution.  It depends are your 
protein system.  A little more biochemistry is needed, but find a nice assay 
system to verify what you are doing.

Good luck,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On Mar 13, 2014, at 6:40 PM, Benini Stefano (P)  wrote:

> Dear All (those dealing with wetlab stuff..),
> 
> While purifying a FAD containing protein we lose part of the FAD (on the gel 
> filtration we clearly see two bands corresponding to holoprotein and free 
> FAD).
> 
> We obtain crystals but diffracting to only about 4 A despite their beautiful 
> look. Our hypothesis is that the crystals contain a population of molecules 
> with and without FAD (?).
> 
> The questions are:
> 
> 1) how to keep FAD bound to the protein during purification and 
> crystallization?
> 
> 2) how to completely remove FAD from the protein? 
> 
> Thank you very much for any help provided!
> 
> Best regards
> 
> Stefano (part-time wetlab person)
> 
> 
> Dr Stefano Benini, Ph.D.
> Assistant Professor
> 
> First International workshop: "Molecular Basis of Fire Blight", Bolzano 
> 15.10.2014
> 
> Laboratory homepage:
> http://pro.unibz.it/staff2/sbenini/B2Cl.htm
> 
> Personal homepage
> http://pro.unibz.it/staff2/sbenini/
> 
> "I don't like anything that's fake and I hate pretenders!"
> 
> *
> Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl)
> Faculty of Science and Technology
> Free University of Bolzano
> Piazza Università, 5
> 39100 Bolzano, Italy
> Office (room K2.14):  +39 0471 017128
> Laboratory (room E.021): +39 0471 017910
> Fax: +39 0471 017009
> 
> "ogni giorno in più è un giorno in meno."

Re: [ccp4bb] ITC with unfolded proteins

[Anita P]

That is a very interesting question, which I would request the seniors out
there to give their insights on.

I was imagining that a recombinant purification of an unfolded partner
would aggregate which would cause trouble in ITC. Am I correct in this
theory?
Would love to have more insights.

thanks in advance
Anita
On Fri, Mar 14, 2014 at 7:18 PM,  wrote:

> Hi,
>
> I think the experiment is doable, but how would you decouple
> protein-protein interaction from folding of the unfolded
> protein due to protein interaction?
>
> Reza
>
> Reza Khayat, PhD
> Assistant Professor
> The City College of New York
> Department of Chemistry, MR-1135
> 160 Convent Avenue
> New York, NY  10031
> Tel. (212) 650-6070
>
>
>  Original message 
> >Date: Fri, 14 Mar 2014 18:07:48 +0530
> >From: CCP4 bulletin board  (on behalf
> of Anita P )
> >Subject: [ccp4bb] ITC with unfolded proteins
> >To: CCP4BB@JISCMAIL.AC.UK
> >
> >   Hello everyone,
> >   I have a query for the scientists working on
> >   protein-protein interaction.
> >   It is known that some proteins exist in unfolded or
> >   molten globule state and attain structure on
> >   interaction with other folded proteins.
> >   Many a times, it is difficult to obtain the
> >   structure of these complexes.
> >   Is it possible to quantitatively determine the
> >   thermodynamics of interaction between an unfolded
> >   protein and a folded protein using ITC? Later may be
> >   perform an alascan to determine the residues of the
> >   unfolded partner involved in the interaction.
> >   Please share your ideas
> >   cheers**
> >   Anita
>

Re: [ccp4bb] off-topic: protein losing FAD during purification

[Orru, Roberto]

Dear Stefano,

Here few thoughts:
You should calculate the amount of holo/apo protein that you get after the 
column. A ratio 280/450 between 9-12 it will suggest that your protein is 
almost completely in the holo-form. So, I will be worried about the peak of 
flavin only if this ratio become much higher than 12.
If this is the case, I would analyze the flavin peak to evaluate if it is 
really FAD or, for example, FMN. You can do easily with a quick silica TLC. 
This mainly because can happen that during the folding there is a 
misincorporation of the cofactor (I am assuming that you are sure that FAD is 
your cofactor).
Otherwise, if your expression levels are too high, maybe the expression strain 
is not able to produce enough flavins to be incorporated. In this scenario, you 
can try to slow down the induction and/or  use more rich media (like terrific 
broth, where the excess of glycerol can also help in keeping bounded the 
flavin) as add 1-2 mM of riboflavin in the broth.

If this doesn't work, during the purification I would suggest to add some FAD 
in the lysis buffer (~50 uM) and 10% glycerol to increase the viscosity of all 
the buffers and reduce the diffusion. You would maybe need also to play with 
salt concentrations to see if there is an effect in the flavin binding. 
Remember: flavins are also light sensible. Be sure to keep the sample in the 
dark as much as possible during all the steps (i.e. use aluminum foil to wrap 
the column or the tubes where your sample is stored).

To remove FAD there are many protocols that you can follow, but mainly it 
require a dialysis in presence of KBr in a light acidic environment. You can 
find protocols in literature looking for papers by Edmondson, Van Berkel or 
Massey to start or chapter 11 in flavoprotein protocols (Vol 131, methods in 
molecular biology).

Good luck,
Roberto

__
Roberto  Orru, PhD
Department of Chemistry, Emory University
1515 Dickey Drive
Atlanta, GA. 30322 (USA)



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Benini 
Stefano (P)
Sent: 13 March, 2014 18:40
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: protein losing FAD during purification

Dear All (those dealing with wetlab stuff..),

While purifying a FAD containing protein we lose part of the FAD (on the gel 
filtration we clearly see two bands corresponding to holoprotein and free FAD).

We obtain crystals but diffracting to only about 4 A despite their beautiful 
look. Our hypothesis is that the crystals contain a population of molecules 
with and without FAD (?).

The questions are:

1) how to keep FAD bound to the protein during purification and crystallization?

2) how to completely remove FAD from the protein?

Thank you very much for any help provided!

Best regards

Stefano (part-time wetlab person)


Dr Stefano Benini, Ph.D.
Assistant Professor

First International workshop: "Molecular Basis of Fire Blight", Bolzano 
15.10.2014

Laboratory homepage:
http://pro.unibz.it/staff2/sbenini/B2Cl.htm

Personal homepage
http://pro.unibz.it/staff2/sbenini/

"I don't like anything that's fake and I hate pretenders!"

*
Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl) Faculty of 
Science and Technology Free University of Bolzano Piazza Università, 5
39100 Bolzano, Italy
Office (room K2.14):  +39 0471 017128
Laboratory (room E.021): +39 0471 017910
Fax: +39 0471 017009

"ogni giorno in più è un giorno in meno."



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Re: [ccp4bb] ITC with unfolded proteins

[Paula Salgado]

Dear Anita

One alternative method to determine the thermodynamics and potentially discern 
the folding energy changes from the interaction driven ones would be NMR. 
Advantages over ITC experiments include:  determine if the interaction drives 
foldness, estimate associated thermodynamics and, if you do 3D labelling 
experiments and do backbone assignment, you can map the interaction interface 
from the NMR data. Elegant NMR experiments have been extensively exploited to 
study  unfolded/folded states and interactions in recent times, with exciting 
results which proved to be unachievable by any other 
biochemical/biophysical/structural technique, so it was definitely worth trying!

Good luck
Paula


===

Dr Paula S. Salgado
Lecturer in Macromolecular Crystallography
Institute for Cell and Molecular Biosciences
Faculty of Medical Sciences
3rd Floor Cookson Building
Newcastle University
Newcastle upon Tyne, NE2 4HH, UK

Tel: +44 (0)191 222 7369
Fax: +44 (0)191 222 7424
Email: paula.salg...@ncl.ac.uk

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Anita P 
[crystals...@gmail.com]
Sent: 14 March 2014 13:57
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC with unfolded proteins

That is a very interesting question, which I would request the seniors out 
there to give their insights on.

I was imagining that a recombinant purification of an unfolded partner would 
aggregate which would cause trouble in ITC. Am I correct in this theory?
Would love to have more insights.

thanks in advance
Anita
On Fri, Mar 14, 2014 at 7:18 PM, 
mailto:rkha...@ccny.cuny.edu>> wrote:
Hi,

I think the experiment is doable, but how would you decouple
protein-protein interaction from folding of the unfolded
protein due to protein interaction?

Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
>Date: Fri, 14 Mar 2014 18:07:48 +0530
>From: CCP4 bulletin board 
>mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf
of Anita P mailto:crystals...@gmail.com>>)
>Subject: [ccp4bb] ITC with unfolded proteins
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hello everyone,
>   I have a query for the scientists working on
>   protein-protein interaction.
>   It is known that some proteins exist in unfolded or
>   molten globule state and attain structure on
>   interaction with other folded proteins.
>   Many a times, it is difficult to obtain the
>   structure of these complexes.
>   Is it possible to quantitatively determine the
>   thermodynamics of interaction between an unfolded
>   protein and a folded protein using ITC? Later may be
>   perform an alascan to determine the residues of the
>   unfolded partner involved in the interaction.
>   Please share your ideas
>   cheers**
>   Anita

Re: [ccp4bb] ITC with unfolded proteins

[Xiaodi Yu]

I have done once by using two proteins, one is disordered, the other is very 
well folded. The result I got is the baseline drift. The baseline goes up upon 
each injection. The reason I thought at that time is the heat capacity changed 
dramatically in the system. The disordered protein may form some degree of 
structure after interacting with its partner. And we did find some 
precipitation after the experiment.

Dee



Xiaodi.yu@childrens.harvard edu

Sent from my iPad

> On Mar 14, 2014, at 9:57 AM, "Anita P"  wrote:
> 
> That is a very interesting question, which I would request the seniors out 
> there to give their insights on.
> 
> I was imagining that a recombinant purification of an unfolded partner would 
> aggregate which would cause trouble in ITC. Am I correct in this theory?
> Would love to have more insights.
> 
> thanks in advance
> Anita
>> On Fri, Mar 14, 2014 at 7:18 PM,  wrote:
>> Hi,
>> 
>> I think the experiment is doable, but how would you decouple
>> protein-protein interaction from folding of the unfolded
>> protein due to protein interaction?
>> 
>> Reza
>> 
>> Reza Khayat, PhD
>> Assistant Professor
>> The City College of New York
>> Department of Chemistry, MR-1135
>> 160 Convent Avenue
>> New York, NY  10031
>> Tel. (212) 650-6070
>> 
>> 
>>  Original message 
>> >Date: Fri, 14 Mar 2014 18:07:48 +0530
>> >From: CCP4 bulletin board  (on behalf
>> of Anita P )
>> >Subject: [ccp4bb] ITC with unfolded proteins
>> >To: CCP4BB@JISCMAIL.AC.UK
>> >
>> >   Hello everyone,
>> >   I have a query for the scientists working on
>> >   protein-protein interaction.
>> >   It is known that some proteins exist in unfolded or
>> >   molten globule state and attain structure on
>> >   interaction with other folded proteins.
>> >   Many a times, it is difficult to obtain the
>> >   structure of these complexes.
>> >   Is it possible to quantitatively determine the
>> >   thermodynamics of interaction between an unfolded
>> >   protein and a folded protein using ITC? Later may be
>> >   perform an alascan to determine the residues of the
>> >   unfolded partner involved in the interaction.
>> >   Please share your ideas
>> >   cheers**
>> >   Anita
> 

Re: [ccp4bb] ITC with unfolded proteins

[Zhengrong Yang]

One of my main concerns is that the unfolded protein itself would irreversibly 
aggregate and then wouldn't interact with the folded protein. I think DSC 
(differential scanning calorimetry) should be performed first to characterize 
the state of both proteins and their potential "complex". By analyzing the DSC 
data, you should have an idea what kind of enthalpy change is involved during 
the complex formation. If you see the unfolded protein does become folded, then 
ITC experiments are worth trying. A combination of ITC/DSC experiments may help 
you tweak out the exact interaction parameters.

Wendy

--
Wendy Yang
Manager, Biocalorimetry Laboratory
Center for Biophysical Sciences & Engineering
University of Alabama at Birmingham
Tel: 205-975-2450
Fax: 205-934-0480
Email: y...@cbse.uab.edu



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anita P
Sent: Friday, March 14, 2014 8:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC with unfolded proteins

That is a very interesting question, which I would request the seniors out 
there to give their insights on.

I was imagining that a recombinant purification of an unfolded partner would 
aggregate which would cause trouble in ITC. Am I correct in this theory?
Would love to have more insights.

thanks in advance
Anita
On Fri, Mar 14, 2014 at 7:18 PM, 
mailto:rkha...@ccny.cuny.edu>> wrote:
Hi,

I think the experiment is doable, but how would you decouple
protein-protein interaction from folding of the unfolded
protein due to protein interaction?

Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070


 Original message 
>Date: Fri, 14 Mar 2014 18:07:48 +0530
>From: CCP4 bulletin board 
>mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf
of Anita P mailto:crystals...@gmail.com>>)
>Subject: [ccp4bb] ITC with unfolded proteins
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hello everyone,
>   I have a query for the scientists working on
>   protein-protein interaction.
>   It is known that some proteins exist in unfolded or
>   molten globule state and attain structure on
>   interaction with other folded proteins.
>   Many a times, it is difficult to obtain the
>   structure of these complexes.
>   Is it possible to quantitatively determine the
>   thermodynamics of interaction between an unfolded
>   protein and a folded protein using ITC? Later may be
>   perform an alascan to determine the residues of the
>   unfolded partner involved in the interaction.
>   Please share your ideas
>   cheers**
>   Anita

Re: [ccp4bb] twin refinement

[Eleanor Dodson]

If the twin law is k,h,-l, then your a axis must almost equal the b axis?
And if the twin fraction is 0.48 then you have additional symmetry I guess?

How sure are you that the point group is P4/mmm?




On 13 March 2014 20:41, Teresa Swanson  wrote:

> Dear collegues,
>
> I'm working with a drug complexed protein structure that is having major
> twinning issues. The drug has a single Br atom on a benzene ring, which I'd
> like to use for orienting the drug in the binding site.  I have various
> anomalous data sets, ranging from 3.0A resolution, all scaled into P222
> with a Rlin of .125.
>
> Using MR, the twin law (k, h, -l) and NCS restraints, I can confidently
> solve the structure without anomalous, and the drug density is clear in the
> Fo-Fc map, with Rw/Rf at ~.26/.29 and a space group of P21221. It might be
> important to note that any simulated annealing I've tried invariably
> increases the Rfree by 2-3%, so I've scraped it. As you can imagine, when
> using the twinned data, the anomalous maps are weak and random.
>
> I've used the Phenix "detwin" option in Xtriage to see if I can pull the
> anomalous signal out of it. If I use the .mtz file that is output for MR
> and calculate the anomalous maps, it looks promising. The twin fraction for
> the one particular dataset I've been using is estimated at approx .48. Is
> this too close to 50% to do the detwinning? Now I'm wondering how to
> properly refine this further. I'm assuming that since I've "detwinned" the
> data, I do refinement without the twin law. But that gives an initial Rf of
> .38 when using it gives .31.  Since I've already solved the structure
> without using the anomalous flag, can I just use the "detwinned"
> reflections and the refined structure to calculate an anomalous map
> (without having to redo the refinement)?
>
> Mainly, my main question is about how to tease out and properly refine the
> anomalous data from a twinned structure. Also, how much of a difference
> will it make to scale into P222 versus P21212. And, if I have quite high
> redundancy, should I "scale anomalous" in HKL2000 or just use the
> "anomalous" flag?
>
> Any help on refining this twinned structure would be greatly appreciated!
> Thanks,
>
> Teresa
> PhD Student
>

Re: [ccp4bb] ITC with unfolded proteins

[DUMAS Philippe (VIE)]

Le Vendredi 14 Mars 2014 13:37 CET, Anita P  a écrit:

Anita,

If one of the partners  is indeed more or less unfolded before interaction, 
then you should see a negative DeltaS upon complex formation.
Practically, I would try first putting the unfolded protein in the cell and the 
folded one in the syringe in view of possible solubility problems with an 
"incorrectly" folded protein.
Alanine scanning ? Yes on the paper, but may be less feasible in practice due 
to the amount of material to prepare.

Good luck
Philippe Dumas


> Hello everyone,
>
> I have a query for the scientists working on protein-protein interaction.
> It is known that some proteins exist in unfolded or molten globule state
> and attain structure on interaction with other folded proteins.
> Many a times, it is difficult to obtain the structure of these complexes.
>
> Is it possible to quantitatively determine the thermodynamics of
> interaction between an unfolded protein and a folded protein using ITC?
> Later may be perform an alascan to determine the residues of the unfolded
> partner involved in the interaction.
>
> Please share your ideas
>
> cheers
> Anita

Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot

[James Holton]

An Fo-Fc map is actually the real-space representation of the Fo-vs-Fc R 
factor (Rcryst), so the "sigma" of this map will continue to drop 
relative to the "true" electron scale as your model improves and the 
difference between Fo and Fc diminishes. The 2Fo-Fc map, however, is a 
best guess of the "total" electron density, and that is always on about 
the same scale, so the "sigma" of this map is pretty constant throughout 
refinement. Toward the end of refinement, when your R/Rfee are around 
20%, the "sigma" of the Fo-Fc map will be about 1/5 of the 2Fo-Fc 
"sigma" level.  So, eventually you will see things at the "3 sigma" 
level in the Fo-Fc map that are not "visible" at the "1 sigma" contour 
level of a 2Fo-Fc map.  This does not mean they are "noise".  I'm not 
sure where that "rule" came from.  The "noise level" is actually another 
1/5th below the Fo-Fc "sigma" level, or 25-fold smaller than the "1 
sigma" contour of the 2Fo-Fc map.  This is because the "noise" is not 
really related to R/Rfree but rather the actual error in the data.  This 
is roughly half the value of redundancy-corrected Rmerge-like values 
such as Rpim.  The factor of two is because these latter R statistics 
are for intensities and the maps are calculated from amplitudes.


The situation where the "sigma" of the Fo-Fc map is the "noise level" 
really only arises when the difference between Fo and Fc (aka Rcryst) is 
comparable to Rpim, and that pretty much only happens for small 
molecules where all the atoms in the unit cell can be named and 
accounted for.  Macromolecular models generally don't achieve that.  Not 
yet anyway.


So, I encourage you to build into Fo-Fc density if you can. Remember, 
there is always "something" there, the question is what that "something" 
is.  And also "what else could it be"?  The "bunch of waters" model is 
always an alternative hypothesis.  It is fairly easy to show that one 
model fits better than another, but to show that the difference is 
"significant" requires error bars, and that's why we developed the RAPID 
procedure:

http://dx.doi.org/10.1073/pnas.1302823110

-James Holton
MAD Scientist

On 3/10/2014 1:41 AM, herman.schreu...@sanofi.com wrote:


Dear Amlan,

The sigma of an Fo-Fc map map depends on the residual noise in your 
map. In a well-refined structure, the sigma will be low, so at 3 sigma 
it will show very weak features.


My guess is that your ligand is present in partial occupancy and that 
you will find it in your 2Fo-Fc map when you scroll down your contour 
level. If you see convincing Fo-Fc density without a ligand being 
fitted, the presence of the ligand must be real and you can fit it. 
However, I would refine a group occupancy for your ligand.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *Amlan Roychowdhury

*Gesendet:* Montag, 10. März 2014 09:09
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] regarding Fo-Fc map in coot

Dear All,

Some times during model building in coot we have found that at the 
position of ligand molecules and water, there is a good Fo-Fc map 
(above 3 sigma), devoid of any 2Fo-Fc map.


1.What does it physically mean and why the 2Fo-Fc map was not 
generated properly?


2. Can we fit ligand molecule there?

Thanks in advance.

Best Wishes

Amlan.

--
Amlan Roychowdhury.
Senior Research Fellow.
Protein Crystallography Lab.
Dept. of Biotechnology,
IIT Kharagpur.
Kharagpur 721302
West Bengal.
India.

Re: [ccp4bb] twinning problem ?

[Keller, Jacob]

At the limit, the microdomain picture leads to powder-diffraction-type spots 
(rings), provided the block size is relatively large with respect to the unit 
cell. And as the blocks get smaller, the distinction between "changing unit 
cell parameters" and "mosaic block misorientation" dissolves.

I am wondering, then, what one explains by positing microdomains, actually? Is 
there strong evidence supporting their existence?

JPK




-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Nave
Sent: Thursday, March 13, 2014 7:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] twinning problem ?

Hi Zbyszek
I think this has deviated significantly from twinning problems!

I certainly don't claim the 1998 study was typical. The crystal was large by 
present day standards, no cryoprotectant was used and non uniform 
drying/cooling rates might have occurred. 

The Juers et. al. paper includes the statement "However, in most cases [omega] 
does not dominate, suggesting that [delta]a/a plays a significant role in 
nearly all of our samples." 
There is also the Kriminski paper 
(http://journals.iucr.org/d/issues/2002/03/00/en0056/index.html) which includes 
the statement " Flash-cooling tetragonal lysozyme crystals degrades diffraction 
resolution and broadens the distributions of lattice orientations (mosaicity) 
and lattice spacings. The diffraction resolution strongly correlates with the 
width of the lattice-spacing distribution."
The Diedrichs paper includes "The experience of the author is that for most 
protein crystals reflections are not markedly elongated along circles 
corresponding to their d-spacing; therefore, `rotational mosaicity' appears to 
play a minor role . the model calculations suggest that, apart from 
inhomogeneity and disorder in unit cells, unit-cell parameter variations are 
responsible for most of the imperfections that result in poor diffraction 
properties of crystals.

Of course selectively quoting papers can be misleading!

Fig. 5A of Juers et al lumps omega and delta a/a together and does not 
distinguish between the two. The plot is [eta] versus d. The slope of a line 
fit to this plot gives an estimate of 1/s, while the y intercept estimates 
[omega] + [delta]a/a. In this case, s is the mosaic block size.


To summarise cryocooling can produce a fragmentation in to smaller mosaic 
blocks with larger angular variation between blocks and a distribution of cell 
dimensions between blocks and within blocks (elastic strain). It really needs a 
high resolution diffraction set up (to detect diffracted beam divergences above 
those given by the incident beam divergence) to distinguish between the various 
effects. 
Of course, in some cases, such a set up could reveal certain types of twinning 
(so I have left the subject of the email unchanged!)

Regards
  Colin
-Original Message-
From: Zbyszek Otwinowski [mailto:zbys...@work.swmed.edu]
Sent: 13 March 2014 21:33
To: ccp4bb
Subject: Re: [ccp4bb] twinning problem ?

On 03/13/2014 10:55 AM, Keller, Jacob wrote:
>> Unless you are interested in finding curious objects, what would you do with 
>> protein quasicrystal? The practices of macromolecular crystallography is 
>> about determining 3-dimensional structure of objects being crystallized. 
>> Protein quasicrystal are really unlikely to diffract to high enough 
>> resolution, and even ignoring all other practical aspects, like writing 
>> programs to solve such a structure, chances of building an atomic model are 
>> really slim.
>
> Right, if crystallography is seen as purely a tool for biology I agree. As 
> for curious objects, I think almost all profound breakthroughs come from 
> unadulterated curiosity and not desire for some practical end. Not sure why a 
> priori this should be so, but just consider your favorite scientific 
> breakthrough and whether the scientist set out to make the discovery or not. 
> Some are, but most are not, I think. Maybe aperiodic protein crystals have 
> some important function in biology somewhere, or have unforeseen materials 
> science properties, analogous to silk or something.
>
>>> This is easy to test by analyzing diffraction patterns of individual 
>>> crystals.
>> In practice, the dominant contribution to angular broadening of 
>> diffraction peaks is angular disorder of microdomains, particularly in 
>> cryo-cooled crystals.
>> However, exceptions do happen, but these rare situations need to be 
>> handled on case by case basis.
>> The interpretation of the data presented in this article is that variation 
>> in unit cell between microcrystals induce their spatial misalignment. The 
>> data do not show variation of unit cell within individual microscrystalline 
>> domains.
>> Tetragonal lysozyme can adopt quite a few variations of the crystal lattice 
>> during cryocooling. Depending on the conditions used, resulting mosaicity 
>> can vary from 0.1 degree (even for 1mm size crystal) t

Re: [ccp4bb] off-topic: protein losing FAD during purification

[ramesh vandanapu]

Dear All
 On similar lines any suggestions and tips on expression and
purification of the proteins containing both FAD and NADPH would be really
helpful.
I recently tried to express and purify a protein containing these two
ligands, but failed (aggregation of protein).

my initial questions are:
does addition of Riboflavin in the media has affect on protein expression?
considering reconstitution, I am concerned that NADHP will be oxidized
during processing.
what are the other alternatives for incorporating ligands, if
reconstitution doesn't help?

Re: [ccp4bb] twin refinement

[Jon Schuermann]

Hi Teresa,

   As Eleanor has mentioned, you should probably check out other space 
groups. Xtriage gives a lot of great information and many plots to 
inspect. But, if you do not know what the plots mean and just look at 
the results that say the twin fraction is 0.48 you can get into some 
trouble. From what I have seen over the years, when most people say they 
have a twin fraction nearly 0.5, they are usually in the wrong space 
group and the data is not twinned. When the summary table shows a low 'R 
obs' and a high twin fraction across the tests that means that the 
operator is present in the data. But that could be a crystallographic 
operator or a twin operator. You would have to inspect the plots to see 
if the data looks twinned, but if there is pseudosymmetry it could make 
the plots look not twinned. You could run the extra twin tests in 
Xtriage inputting the MR solution so that the R vs. R plot (Andrey 
Lebedev, et al. (2006) , Acta Crystallogr. D62, 83-95) could be 
calculated. In the end, your data could be nearly perfectly twinned, I 
have had it happen a couple of times, but I always tell people to assume 
it is not until the model building is done and the R-factor still won't 
drop. Your R-factors are not that bad for 3A resolution, indicating the 
operator might be crystallographic and you are too low in symmetry.


   So if you have done all this and you still believe the data is 
twinned... Don't detwin your data! Your twin fraction is too high and it 
will introduce a lot of errors. This has been gone over many times and 
you can search the archives. I never detwin data regardless of twin 
fraction. Modern programs like Phenix.refine and Refmac (I think) handle 
twinned data just fine. If you are just trying to calculate an ADF map 
to find the Br then it might be difficult since the peak heights are 
going to be lower. Your best bet is to increase your redundancy without 
killing the crystal with too much radiation damage. If your redundancy 
is around 10 or so then you could probably use the 'scale anomalous' 
option. I tend to scale with and without it and look at the maps to see 
if it helps or not. Sometimes it helps, other times it doesn't and I am 
sure there are reasons (like anisotropy). Another word of caution is the 
possibility of model bias in the maps when the twin fraction approaches 
0.5. The programs will use the model in the detwinning process if the 
twin fraction is above a certain threshold. I believe it is 0.45 in 
Phenix.refine.


First thing I would do is follow Eleanor's lead and check out other 
space groups. You might have pseudo-symmetry with or without twinning.


Jon

--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist, NE-CAT
Argonne National Laboratory, 436E
9700 S. Cass Ave.
Argonne, IL 60439

Email: schue...@anl.gov
Tel: (630) 252-0682


On 03/13/2014 03:41 PM, Teresa Swanson wrote:

Dear collegues,

I'm working with a drug complexed protein structure that is having major 
twinning issues. The drug has a single Br atom on a benzene ring, which I'd 
like to use for orienting the drug in the binding site.  I have various 
anomalous data sets, ranging from 3.0A resolution, all scaled into P222 with a 
Rlin of .125.

Using MR, the twin law (k, h, -l) and NCS restraints, I can confidently solve 
the structure without anomalous, and the drug density is clear in the Fo-Fc 
map, with Rw/Rf at ~.26/.29 and a space group of P21221. It might be important 
to note that any simulated annealing I've tried invariably increases the Rfree 
by 2-3%, so I've scraped it. As you can imagine, when using the twinned data, 
the anomalous maps are weak and random.

I've used the Phenix "detwin" option in Xtriage to see if I can pull the anomalous signal out of 
it. If I use the .mtz file that is output for MR and calculate the anomalous maps, it looks promising. The 
twin fraction for the one particular dataset I've been using is estimated at approx .48. Is this too close to 
50% to do the detwinning? Now I'm wondering how to properly refine this further. I'm assuming that since I've 
"detwinned" the data, I do refinement without the twin law. But that gives an initial Rf of .38 
when using it gives .31.  Since I've already solved the structure without using the anomalous flag, can I 
just use the "detwinned" reflections and the refined structure to calculate an anomalous map 
(without having to redo the refinement)?

Mainly, my main question is about how to tease out and properly refine the anomalous data from a 
twinned structure. Also, how much of a difference will it make to scale into P222 versus P21212. 
And, if I have quite high redundancy, should I "scale anomalous" in HKL2000 or just use 
the "anomalous" flag?

Any help on refining this twinned structure would be greatly appreciated!
Thanks,

Teresa
PhD Student



--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist, NE-CAT
Argonne National Laboratory, 436E
9700 S. Cass Ave.
Argonne, IL 60439

Email: schue...@anl.