Re: [ccp4bb] molprobity clashscore, symmetry-related molecules?

2014-10-24 Thread Nadir T. Mrabet

MolProbity works on chains present in the pdb file.
Therefore, I would predict that if the pdb file can be made to consist 
of several chains (built by symmetry operations) and bearing each a 
distinct chain name, then MolProbity would (artificially) work on 
symmetry-related molecules as well.


Nadir

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
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On 23/10/2014 13:06, "Oliver Smart" wrote:

on 23/10/14 11:52 AM, Tim  wrote:


Hi everybody,
Molprobity does not take into account contacts/clashes from
symmetry-related molecules, or does it?
Thanks in advance,
Tim


Tim,

I am not sure. In my experience MolProbity reduce does not take crystal
contacts
into account (but reduce does a great job otherwise). But this might have
been improved.

Oliver
-
Dr Oliver Smart

Director SmartSci Limited  http://www.smartsci.uk/
& Consultant Global Phasing Ltd http://www.globalphasing.com/



Re: [ccp4bb] [Proteopedia] announcement: course on Proteopedia with JSmol

2014-09-23 Thread Nadir T. Mrabet

Dear Angel, Jaime and Joel,

Proteopedia is great work, but I am unlucky as I won't be able to visit 
you at Alcalà.
This makes me think it would be great to have a web seminar on the 
topic. Thanks, Jaime!


Enjoy your meeting in Madrid and around.

Best regards,

Nadir

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
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On 23/09/2014 19:17, Joel Sussman wrote:

Dear Jmol aficionados,

For those to happen to be in Spain (or able to come, you will be most
welcome), I'd like to share the information about a 2-day course that
we will held in Alcalá thanks to a visit of Jaime Prilusky.

Dates are October 2nd and 3rd (Thu-Fri), in my University (Alcalá de
Henares, in Madrid province)

The course will focus on Proteopedia and its uses to study, display
and teach macromolecules. It will be run in English and/or Spanish,
according to the audience.

All information is available at http://bit.ly/P14UAH

Hope to see some of you!


Dr. Angel Herráez
Biochemistry and Molecular Biology,
Dept. of Systems Biology, University of Alcalá
E-28871 Alcalá de Henares  (Madrid), Spain





Re: [ccp4bb] Guard columns from FPLC

2014-09-16 Thread Nadir T. Mrabet
Well, Pharmacia (now, GE) used to sell such a guard column for FPLC 
prior to Akta.
And I remember buying adaptors to be able to connect the guard column 
and FPLC columns to an HPLC system.

So all the tools exist.
But keep in mind that a guard column volume must in all cases be kept 
minimal to avoid sample dilution which can be deleterious to resolution 
in size-exclusion chromatography.
Prior to FPLC with (or without) a guard column, I have always used an 
Eppendorf microcentrifuge followed by 2u-filtering prior to injecting 
the sample and have consistently observed that these remove most 
aggregates if not all.

HTH,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
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On 16/09/2014 19:01, Christian Roth wrote:

Hi Anita,

never heared about Guard columns for Superdex columns. These guard 
columns are usually for HPLC columns (RP) or Silica High pressure 
gelfiltration columns like from Tosoh Bioscience and part of the 
acutal column itself, though replaceable.


Christian

Am 16.09.2014 09:29, schrieb Anita P:

Hi All,
Sorry for this off topic.

I have heard that there are these little columns called guard columns
which can be attached to AKTA purifiers. These columns prevent the
incoming huge aggregates to be deposited and blocking of the gel
filtration columns.

Can any one advice me regarding where to purchase these columns. I could
not find them in GE website. We have Superdex 16/60 on AKTA purifier.

Thanks in advance.
Have a good day

Anita




Re: [ccp4bb] areaimol and hydrogens

2014-07-28 Thread Nadir T. Mrabet

Jose,
Your are most probably right.
Atoms used for ASA calculations are "unified atoms" as their vdW radii 
incorporate light atoms (hydrogens) which, by and large, 
crystallographers don't see.

Adding extra H atoms is likely to end up in miscalculations.
Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
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On 28/07/2014 14:20, Jose Manuel Duarte wrote:
I believe that ignoring hydrogen atoms is the default behaviour of any 
software calculating ASA values. Normally the VdW radii values of the 
heavy atoms already include the hydrogens implicitly.


If your input structure has hydrogens and they were included in the 
calculation it would result in over-estimating the ASA values. I'm not 
familiar with AREAIMOL but I guess it must behave in a similar way.


Jose



On 28/07/14 14:02, Harry Mark Greenblatt wrote:

BS"D

Dear All,

   I understood from the areaimol documentation that hydrogens are 
not included as one of the default atoms.  But one can add atoms, and 
so I added an "ATOM" line for hydrogen.  The program quite happily 
accepted my input line, but later on stated explicitly that it was 
ignoring the hydrogens.  Is there no way to include hydrogens?


Thanks

Harry


-

Harry M. Greenblatt

Associate Staff Scientist

Dept of Structural Biology

Weizmann Institute of SciencePhone:972-8-934-3625

234 Herzl St.Facsimile: 972-8-934-4159

Rehovot, 76100

Israel


harry.greenbl...@weizmann.ac.il <mailto:harry.greenbl...@weizmann.ac.il>














Re: [ccp4bb] metal chelation

2014-05-19 Thread Nadir T. Mrabet

Hi Adam,
I have not read all the thread as it came all at once and late (9:00pm 
here).
I believe the best way to strip a protein of metals is to first adsorb 
it onto a solid support (e.g. IEX) and then use a sufficiently low-pH 
(say equal or below 6) buffer that contains also EDTA.

You will probably need several washes but it works!
Also be aware that EDTA binds well to several proteins.
HTH,
Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
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On 19/05/2014 20:21, Adam Brummett wrote:
Thank you everyone for the comments and suggestions. To answer a few 
questions:


-I do not use a treated buffer system. I have just used the nano-pure 
water. I have looked into Chelex, but before I bought it I wanted to 
see if you all recommended it. I was trying to avoid this, but it may 
not be possible now.


-the active site does bind metals and is promiscuous in binding, so it 
is not know if the His tag or active is the source of contamination, 
but cleavage is not an option for us. The biding of metal is going to 
be needed for phasing, so good point Tim, hopefully just not in the 
His site.


-thank you Vivoli for the protocol, seems very thorough. Have you had 
success with it? I anticipate I'll need to go down this road.


-Roger, the metals you mentioned (Zn and Fe) are the problem and I 
expect to have to go to heroic measures to get an apo enzyme . But you 
did mention easier ways of getting metal substituted. I have some 
evidence that I can do this. Do you have any other thoughts on this 
matter? Maybe a reference to something similar (non-apo but could 
substitute?


Thank you all so much for the help and advice.

-Adam


On May 19, 2014, at 12:55 PM, Roger Rowlett <mailto:rrowl...@colgate.edu>> wrote:



The answer depends on a number of questions:

  * What metal ion are you trying to eliminate?
  * What kind of metal-binding site is involved?
  o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
  o An active site coordinated metal? (e.g.,
metalloenzyme)--these can be refractory

Many metalloenzymes are not going to give up their metal to 
chelators, or just any chelator, or at all. Denaturation, dialysis, 
and refolding is an extreme way of removing metal ions to make 
apoprotein. Won't work for every protein. Chelation can be highly 
specific, that is one chelator may work, while another, similar one, 
will not.


Some metal ions are notoriously difficult to eliminate, because they 
are adventitious trace contaminants in nearly everything, e.g. zinc 
and maybe even iron. (Plastic-ware seems to be often loaded with 
trace iron, and also is capable of adsorbing metal ions form 
solution.) To make apo-enzymes from zinc proteins, you have to go to 
heroic efforts to ensure that glassware, water, buffers, and reagents 
are zinc-free, especially if you don't have high (mM) concentrations 
of protein to work with.


A His-tag is very likely to snag adventitious metals from solution, 
and can often mess up metal analysis for metalloproteins by providing 
"extra" metal. If this is a problem for your application, you may 
want to consider removing the His-tag.


If you are making apoenzyme to get a different metal installed 
(metallosubstitution), there are slightly easier ways to do that than 
going through the apoenzyme route.


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline 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

On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote:

Hello All,

   I apologize for the non-crystal related question. I am trying to get a fully 
metal-free apo enzyme. The 6x His construct is consistently purified with some 
metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA 
and DFO and then dialyzing it away, but this has shown little to no effect. Any 
thoughts or recommendations would be greatly appreciated. Thanks.

Adam






Re: [ccp4bb] repulsive effects of arginine

2013-10-09 Thread Nadir T. Mrabet
Resonance was to be understood exactly as meaning all the bonds are 
averaged between both types (single and double bonds).
Furthermore, fluctuations in the immediate environment will affect 
electron distribution with time, as proteins exist in a dynamic state.


Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 08/10/2013 19:27, Andrew Purkiss wrote:

An example of pi-pi stacking of the guanidinium groups, can be seen on a
structure which I worked on; pdb-code: 2x2u. Look at the interactions
between Arg 77 and its symmetry mate, with Arg 144 (and symmetry copy)
flanking, giving rise to a stack of 4 Arginine guanidinium groups, with
a sulphate ion neutralising the environment.

Such pi-pi stacking is also commonly seen with Tyrosine and
Phenylalanine, with cation-pi interactions also common (e.g. Lys NZ to
the planar side of Phe). Resonance is not really a correct description
of these delocalised pi-orbitals; as there are no single and double
bonds, but all the bonds are an average of both types.

Additionally, remember that the Arginine(s) may not actually be charged,
as the local environment of ionic sidechains can move the pKa value(s) a
long way from the expected value for an isolated sidechain, with the pH
of the crystallisation condition also potentially affecting what is
charged.

Andrew Purkiss.

On Tue, 2013-10-08 at 18:34 +0200, Nadir T. Mrabet wrote:

Yes, indeed Andrey.
And this results from resonance (tautomerization) of the guanidinium group.

Regards,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 08/10/2013 18:01, Andrey Feklistov wrote:

Hi Jan,

please note, Arg-Arg proximity is not always repulsive: guanidinium groups can 
associate bridged by H-bonds and interactions with water molecules or 
neighboring amino acids. There are many examples of these unusual Arg 
formations, see for reference:

Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein Function 
and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116, 7006−7013

Hope this is helpful,

Andrey



Re: [ccp4bb] repulsive effects of arginine

2013-10-08 Thread Nadir T. Mrabet

Jan,

Ionic interaction does reduce the charges born by the partners in isolation.
This is why charged residues found in the protein core are always paired.
Furthermore, concerning arg, beyond the fact that they are found to 
autointeract as Andrey pointed out, the charge they bear is spread over 
all the guanidinium group so that it is not punctual as is the case for 
lys NZ.

H-bonds also solvate charges.

Are you stating that asp can in no way be entrapped between the arginines?
Recall arg-asp interactions in proteins are "hot spots".

Cheers,

Nadir

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 08/10/2013 18:33, Jan van Agthoven wrote:

Thanks for your responses,

Andrey, I had no idea about these arginine associations. In this case
the arginines are facing each other guanidinium to guanidinium. I
guess they wouldn't attract. Nadir, the asp is not entrapped between
the two arginines. But Hermann is probably right by saying that the
asp is too close to the antibody arginine.

Our binding essays were done by fluorescent labeling of the ligand.
Clustering shouldn't play a role. It's hard to explain, there is no
steric hindrance. I guess I'll have to drop this idea of repulsion.

2013/10/8, Andrey Feklistov :

Hi Jan,

please note, Arg-Arg proximity is not always repulsive: guanidinium groups
can associate bridged by H-bonds and interactions with water molecules or
neighboring amino acids. There are many examples of these unusual Arg
formations, see for reference:

Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein
Function and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116,
7006-7013

Hope this is helpful,

Andrey




Re: [ccp4bb] repulsive effects of arginine

2013-10-08 Thread Nadir T. Mrabet

Yes, indeed Andrey.
And this results from resonance (tautomerization) of the guanidinium group.

Regards,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 08/10/2013 18:01, Andrey Feklistov wrote:

Hi Jan,

please note, Arg-Arg proximity is not always repulsive: guanidinium groups can 
associate bridged by H-bonds and interactions with water molecules or 
neighboring amino acids. There are many examples of these unusual Arg 
formations, see for reference:

Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein Function 
and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116, 7006−7013

Hope this is helpful,

Andrey



Re: [ccp4bb] AW: [ccp4bb] repulsive effects of arginine

2013-10-08 Thread Nadir T. Mrabet

Dear Jan,

Is there a possibility for bifurcated salt bridges/H-bonds where the Asp 
would be entrapped between the two arginines, in which case, repulsive 
effects would not take place ?


Cheers,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 08/10/2013 17:00, Jan van Agthoven wrote:

Dear Herman,

The arginine of the antibody is approximately at same distance from
the arginine of the ligand then the aspartic acid of the receptor,
respectively 3.3 and 3.25 A. But you're right! This same aspartic acid
makes a salt bridge with the arginine of the ligand, when this one is
bound to the receptor. So presumably, the ligand loses one salt bridge
when the antibody binds to the ligand receptor complex.

What is troubling is that the ligand is bound to the receptor by many
other hydrogen bonds and salt bridges. Also, mutation of this aspartic
acid to alanine hasn't shown a dramatic effect on ligand binding. So I
was thinking about a double effect: repulsion +  loss of one salt
bridge.

Best,
Jan


2013/10/8, herman.schreu...@sanofi.com :

Dear Jan,
since electrostatics go with one over distance-square, there may still be
some electrostatic repulsion if the aspartic acid is further away as the
arginine. Another question is, what happens with the arginine of the ligand
in absence of the antibody? Does it then make a salt bridge with the
aspartic acid of the receptor? I expect that losing an a salt-bridge
interaction between ligand and receptor will cause a significant drop in
affinity which may explain the effect of the antibody.

Best,
Herman



-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan
van Agthoven
Gesendet: Montag, 7. Oktober 2013 22:47
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] repulsive effects of arginine

Hi everyone,
I'm working on structure of an antibody that inhibits a receptor. The
antibody doesn't induce any conformational change in the receptor and
doesn't bind the ligand binding site. If we superimpose the receptor with
antibody and ligand the only hindrance we find is a electrostatic repulsion
between two arginines (3.3A): one is part of the antibody and one is part of
the ligand and involved in ligand binding. However the arginine coming from
the antibody makes a salt bridge with an aspartic acid from the receptor.
Does this neutralize it's charge? Can we still say that it has a repulsive
effect?
Thanks



Re: [ccp4bb] Modelling Software for beta turn design

2013-04-24 Thread Nadir T. Mrabet

Hi Wenzong,

I would certainly try it also another way.
Choose you beta turn sequence. Insert it between the sequences of both 
beta stands and submit the whole sequence (beta-turn-beta) to homology 
modeling.

Try every beta turn sequence to select top results.
You could download MolIde (http://dunbrack.fccc.edu/molide/) and do the 
exercise in local mode.

Best  of luck,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr
Cell.: +33 (0)6.11.35.69.09

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On 23/04/2013 00:48, Wenzong Li wrote:

Dear All,

I want to use modelling software to design a tight beta turn replacing a loop 
between two beta strands. Can anyone suggest a program to do this sort of 
modelling?

Thank you
Wenzong



[ccp4bb] Recent IUPAC definition of the hydrogen bond

2013-02-11 Thread Nadir T. Mrabet

Hi,

I wonder how much of an impact should there be on structure refinement, 
validation and, last but not least, molecular modeling, if we take into 
account the recent (2011) re-definition of the hydrogen bond made by the 
IUPAC.


Ref:
"Elangannan Arunan, Gautam R. Desiraju, Roger A. Klein,
Joanna Sadlej, Steve Scheiner, Ibon Alkorta, David C. Clary,
Robert H. Crabtree, Joseph J. Dannenberg, Pavel Hobza,
Henrik G. Kjaergaard, Anthony C. Legon, Benedetta Mennucci,
and David J. Nesbitt
Pure Appl. Chem., Vol. 83, No. 8, pp. 1619–1636, 2011.
doi:10.1351/PAC-REP-10-01-01
© 2011 IUPAC, Publication date (Web): 8 July 2011
Defining the hydrogen bond: An account (IUPAC Technical Report)*"


Thanks for your input and interest.

Greetings,

Nadir Mrabet

--
Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr


Re: [ccp4bb] Off Topic- Cystine Detection

2013-02-07 Thread Nadir T. Mrabet
Perhaps you should have a look at 
http://193.146.160.29/gtb/sod/usu/$UBUG/repositorio/10320076_Hawkins.pdf.
and at "An introduction to methods for analyzing thiols and disulfides: 
Reactions, reagents, and practical considerations. Rosa E. Hansen 1, 
Jakob R. Winther Analytical Biochemistry 394 (2009) 147–158.

HTH,

Nadir Mrabet

Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  univ-lorraine.fr

On 07/02/2013 04:17, Dr. Anthony Addlagatta wrote:

***
This message has been scanned by the InterScan for CSC SSM by IICT security 
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***


Dear Yuri,

If you have access to mass spec, this should be a straight forward experiment. 
Find the
reference here.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291582/pdf/v010p00017.pdf

What was the result in the Ellman´s reaction? Unless you have other reactive 
cysteines
in your protein protein, you should not see any color if the pair of cysteines 
on
surface form disulfide.


Anthony

-
Dr. Anthony Addlagatta
Center for Chemical Biology
Indian Institute of Chemical Technology [IICT]
Tarnaka, Hyderabad
AP-500 607, INDIA
Tel:91-40-27191812
Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1

-- Original Message ---
From: Yuri Pompeu 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 6 Feb 2013 16:10:35 +
Subject: [ccp4bb] Off Topic- Cystine Detection


***
This message has been scanned by the InterScan for CSC SSM at IICT and found to 
be

free of known security risks.

***

Dear All,
I am trying to probe the existence of a disulfide bond on the surface of my 
protein.
I have attempted Ellman´s and my results were not as clear as I would have 
hoped for.
I am not a sulfur/cysteine chemist and would appreciate the advice on what 
experiments

to try!

Thanks a bunch
YAP

--- End of Original Message ---

This Mail Scanned by ClamAV and Spammassassin



Re: [ccp4bb] X-PLOR

2012-05-04 Thread Nadir T. Mrabet

Thanks Francisco.

Nicolas, thanks also for your feedback.

In fact, I had already downloaded the X-PLOR manual, but I needed some 
more details on the H-atoms optimization procedure.

That was for the purpose of a teaching work on a 1995 paper.

Best regards,

Nadir


Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr


On 27/04/2012 16:59, Francisco Hernandez-Guzman wrote:

Nadir,

There is an explicit bulletin board for questions regarding CNS and XPLOR. I 
would suggest posting your question there.

http://tech.dir.groups.yahoo.com/group/cnsbb/

Cheers,

Francisco

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nadir T. 
Mrabet
Sent: Friday, April 27, 2012 6:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] X-PLOR

Hi,

Could someone explain to me the scientific details of the protocols used in 
X-PLOR to (1) build explicit hydrogen atoms onto X-ray structures and
(2) optimize their positions?

Many thanks in advance.

Greetings,

Nadir



[ccp4bb] X-PLOR

2012-04-27 Thread Nadir T. Mrabet

Hi,

Could someone explain to me the scientific details of the protocols used 
in X-PLOR to (1) build explicit hydrogen atoms onto X-ray structures and 
(2) optimize their positions?


Many thanks in advance.

Greetings,

Nadir

--

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr


Re: [ccp4bb] protein structure for high schoolers

2012-01-23 Thread Nadir T. Mrabet
I would suggest MolProbity along with KiNG (on Firefox!) would be most 
appropriate (http://kinemage.biochem.duke.edu/).


HTH,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
N-gere - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr


On 20/01/2012 22:41, James Whittle wrote:

Hi-

I am trying to help my former chemistry teacher set up a demonstration 
of protein structure for her class. I'd like to include electron 
density maps, and maybe show an enzyme active site. Are there 
suggestions from the BB on the easiest way to do this? Would pymol be 
the program of choice, or is there a simpler program that could show 
electron density? Has anyone already created such a demonstration they 
could and have advice on it?


James


Re: [ccp4bb] Metal won't strip from IMAC

2012-01-13 Thread Nadir T. Mrabet

Have run into a similar problem.
Cleared the background color by running 2M NaOH together with 0.2M EDTA.
Better replace BMT with TCEP (1 mM).
Also keep in mind that adsorption is pH dependent, that is the higher 
the pH, the  better is adsorption.

Many proteins adsorb irreversibly above pH 7.0.
If you reduce the pH, say to 5.2-5.5, not only you make adsorption less 
stronger (hence, column capacity may drop down), but you will at the 
same time prevent cysteine oxidation.
You can also increase [imidazole] in the equilibration buffer to reduce 
adsorption.

HTH,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr


On 13/01/2012 01:42, Michael Thompson wrote:

Katherine,

You are not alone. I have inadvertently destroyed a GE HisTrap column with high 
concentrations of proteins that contain many exposed cysteines. In my case the 
Co2+ resin turned a very dark purplish-brown and the protein appeared to have 
crashed out on the column. I didn't try to strip it, because I figured it was 
done for anyway, so I can't tell you any more about the problem. Here's how I 
explained it to myself (whether or not this is actually right I'm not 100% 
sure, but it makes sense in my head). The columns I was using have a maximum 
concentration of 5mM for DTT and 10mM for B-mercaptoethanol. So that seems like 
the column can handle 10mM thiol groups. If you have a protein with many 
cysteines and it is very highly concentrated (as was the case for me) then you 
are adding considerably more thiol groups to the solution. This abundance of 
thiols reduces the metal on the column, and disaster ensues. For me, repeating 
the same prep with less DTT (3mM vs. 5mM) in the buffer fixed the issue. If you 
are concerned about your protein oxidizing at lower concentrations of DTT or 
BME, the other alternative is to switch to TCEP. The IMAC columns can tolerate 
higher concentrations of TCEP, and it is a far superior reducing agent (more 
stable, more reductive, etc.)...but also a lot more expensive (although you can 
get away with using much less because it works so much better).

HTH,

Mike


- Original Message -
From: "Katherine Sippel"
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, January 12, 2012 4:01:10 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Metal won't strip from IMAC

Hi all,

I've run into a bit of a protein purification conundrum and wondered if anyone 
had encountered a similar situation. I've exercised all of my google-fu and 
can't find anything. It's a fairly straightforward setup; His-tagged protein 
and Talon Co2+ resin, load lysate, wash with 5 mM imidazole, elute with 150 mM 
imidazole. There is protein in the elution fractions as would be expected. The 
strangeness occurs when I try to regenerate the column. Using the standard 
protocol of 25 mM MES, 100 mM NaCl pH 5 doesn't change the color of the resin 
back to light pink the way it should with a regenerated column. I try stripping 
with the suggested 0.2M EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 
4% CHAPS and then EDTA, still pink, 1 M NaOH then EDTA, still pink. I've 
checked the resin using a Western (with a really specific monoclonal Ab) and it 
seems that my protein has somehow irreversibly bound to the column and is 
preventing the metal from releasing the sepharose. I've even tried competing 
the protein off with excess Co2+ and Mg2+ (the endogenous divalent bound 
cation).

Clearly the solution is swapping to a Ni column, but this is really bugging me 
now. Has anyone run into this problem with IMAC before?

Background: The protein does bind divalent cations (Mg and Mn) with low 
affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues 
total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers.

Thanks,

Katherine



Re: [ccp4bb] How to assess geometry in a model?

2011-12-09 Thread Nadir T. Mrabet

Along you can consider also using MolProbity online.
Best,
Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr
Cell.: +33 (0)6.11.35.69.09



On 09/12/2011 06:52, Robbie Joosten wrote:

Hi Matt,

WHAT_CHECK writes out a file called check.db that contains per-residue 
scores for several quality metrics. It is fairly easy to parse.


Cheers,
Robbie


Date: Thu, 8 Dec 2011 23:08:45 -0500
From: mattw...@gmail.com
Subject: [ccp4bb] How to assess geometry in a model?
To: CCP4BB@JISCMAIL.AC.UK

Hi Folks

I'm looking for a way to score each atom (or residue) in a model based 
on it's geometry.  I know these scores exists because various software 
packages speak of outliers, even including a sigma value in some cases.


So I'm looking for a simple way to get a complete list (not just 
outliers).  Does anyone know of a package that can be made to output 
these scores?


Thanks,

Matt


Re: [ccp4bb] Description of a newly solved structure

2011-12-01 Thread Nadir T. Mrabet

I would also suggest using MolProbity to assess structure quality.

Best,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 01/12/2011 16:34, Katherine Sippel wrote:
For drawing a 2D fold topology I'd suggest TopDraw 
(http://www.ccp4.ac.uk/html/topdraw.html). To see if you structure 
looks like other structures I'd look at the DALI server 
(http://ekhidna.biocenter.helsinki.fi/dali_server/). Dali is pretty 
good for digging out results/discussion fodder because it gives you 
another structure(s) to compare. PISA would be my suggestion for 
roughly identifying any oligimerization though it's best to back those 
claims up with experimental data. If you've got ligands bound Gerard 
Kleywegt and the PDBe staff have put together a lovely means of 
searching and representing motifs which is also useful for comparative 
purposes (http://www.ebi.ac.uk/pdbe-site/pdbemotif/). There are a 
panoply of good figure generating softwares available but my personal 
preference is Pymol for it's flexibility, comprehensive wiki, and 
supportive users groups (http://www.pymol.org/).


If your just looking for a description of what to put into a results 
and discussion section for a new structure you might try browsing 
through the Acta Cryst D archives for some good examples.


Hope this helps,

Katherine

On Wed, Nov 30, 2011 at 10:49 PM, sadaf iqbal <mailto:sadaf_che...@yahoo.com>> wrote:


Hello everyone,

I have submitted one cysteine protease structure in PDB and now i
have to describe the structure completely for my PhD thesis. I
know some web servers who are good to provide knowledge about
protein but i would like to ask expert persons of this field as i
am quite new in describing a structure. Which softwares/web
servers you prefer when you are going to describe one complete
native structure? I am a chemist basically.

Thanks in advance
Sadaf Iqbal
PhD Scholar
ICCBS, University of Karachi, Pakistan.
& Visiting Scientist
University of Hamburg, Germany.




[ccp4bb]

2011-11-25 Thread Nadir T. Mrabet

Hi,

We have been receiving several spam mail from this person.
Any way to stop that quickly?
Thanks,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 25/11/2011 09:22, Sampath Natarajan wrote:

http://zabara.com.br/jogosonline/include/libs/smarty/templates_c/rpdmgla.htm



Re: [ccp4bb] projecting SC value on protein surface

2011-09-07 Thread Nadir T. Mrabet

Hi Pavlina,

I suggest you have a look at 
http://wiki.c2b2.columbia.edu/honiglab_public/index.php/Software:GRASP2.

Runs on Windows.

HTH

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 07/09/2011 15:57, Pavlina Rezacova wrote:

Dear colleagues,
we would like to project shape complementarity value (calulated in SC 
in ccp4) onto a protein surface.
By reading the manual I learned that it can be done with GRASP 
surface. However we have no acess to GRASP (it only runs on Silicon 
Graphics right?).

Is there any alternative way to do it?
Thanks for any advices and suggestions.
Pavlina



Re: [ccp4bb] off-topic: 2 peaks on Cation

2011-02-21 Thread Nadir T. Mrabet
In your original mail (2011-02-18; 18:45 GMT), you wrote: "Possibly 
deamidation of the protein, in particluar one or more lysines"

Hence, my comment was based on your own writing, that is deamiDation.
Wishing you a better week.

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 18/02/2011 21:07, Soisson, Stephen M wrote:

I'll swap you for the deamination, as that pertains to lysines...it's
been a long week :)

Steve

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Nadir T. Mrabet
Sent: Friday, February 18, 2011 2:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: 2 peaks on Cation

Typo! I actually meant deamidation.

Nadir

Pr. Nadir T. Mrabet
Structural&   Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet   medecine.uhp-nancy.fr



On 18/02/2011 20:36, Christian Roth wrote:

Hi,
you did not mention anything about your protein, but if it shows a

metal

dependency, than different amounts of the metal ion might changen the

overall

charge and influence the interactions with the ion exchanger. There

might be

also a modification of an aminoacid f.e. decarboxylation of an

aspartate or

glutamate.

Christian

Am Freitag 18 Februar 2011 18:13:36 schrieb Ulli Hain:

Hi, I was wondering if anyone had possible explanations for a
   recombinantly expressed soluble protein that runs as 2 equal,

slightly

   overlapping peaks on a cation exhanger but as one peak on a size

exclusion

column and same electrophoretic mobility on SDS-PAGE. -Ulli

Adelaide Ulricke Hain
PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry and Molecular Biology
615 North Wolfe Street
Baltimore, MD  21205


Notice:  This e-mail message, together with any attachments, contains
information of Merck&  Co., Inc. (One Merck Drive, Whitehouse Station,
New Jersey, USA 08889), and/or its affiliates Direct contact information
for affiliates is available at
http://www.merck.com/contact/contacts.html) that may be confidential,
proprietary copyrighted and/or legally privileged. It is intended solely
for the use of the individual or entity named on this message. If you are
not the intended recipient, and have received this message in error,
please notify us immediately by reply e-mail and then delete it from
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Re: [ccp4bb] off-topic: 2 peaks on Cation

2011-02-18 Thread Nadir T. Mrabet

Typo! I actually meant deamidation.

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 18/02/2011 20:36, Christian Roth wrote:

Hi,
you did not mention anything about your protein, but if it shows a metal
dependency, than different amounts of the metal ion might changen the overall
charge and influence the interactions with the ion exchanger. There might be
also a modification of an aminoacid f.e. decarboxylation of an aspartate or
glutamate.

Christian

Am Freitag 18 Februar 2011 18:13:36 schrieb Ulli Hain:

Hi, I was wondering if anyone had possible explanations for a
  recombinantly expressed soluble protein that runs as 2 equal, slightly
  overlapping peaks on a cation exhanger but as one peak on a size exclusion
   column and same electrophoretic mobility on SDS-PAGE. -Ulli

Adelaide Ulricke Hain
PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry and Molecular Biology
615 North Wolfe Street
Baltimore, MD  21205



Re: [ccp4bb] off-topic: 2 peaks on Cation

2011-02-18 Thread Nadir T. Mrabet

Given no info on the protein, it can be anything.
Is it recombinant? Which host? etc.

Oxydation (cys, met) is also a possibility
By the way, deamination concerns asn and gln, not lys.

Best,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 18/02/2011 19:45, Soisson, Stephen M wrote:
Possibly deamidation of the protein, in particluar one or more 
lysines.  What does the Mass spec look like?

Cheers,
Steve


*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *Ulli Hain

*Sent:* Friday, February 18, 2011 12:14 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] off-topic: 2 peaks on Cation

Hi, I was wondering if anyone had possible explanations for a 
recombinantly expressed soluble protein that runs as 2 equal, slightly 
overlapping peaks on a cation exhanger but as one peak on a size 
exclusion column and same electrophoretic mobility on SDS-PAGE.

-Ulli


Adelaide Ulricke Hain
PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry and Molecular Biology
615 North Wolfe Street
Baltimore, MD  21205
Notice:  This e-mail message, together with any attachments, contains
information of Merck&  Co., Inc. (One Merck Drive, Whitehouse Station,
New Jersey, USA 08889), and/or its affiliates Direct contact information
for affiliates is available at
http://www.merck.com/contact/contacts.html) that may be confidential,
proprietary copyrighted and/or legally privileged. It is intended solely
for the use of the individual or entity named on this message. If you are
not the intended recipient, and have received this message in error,
please notify us immediately by reply e-mail and then delete it from
your system.


Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?

2011-01-19 Thread Nadir T. Mrabet
It can go both ways: If you increase atom radii by adding that of the 
probe (e.g. 1.4 A° for a water probe) and calculate the "molecular 
surface" using a zero probe with atom radii as previously defined (Ri 
+1.4 A°), the area you get is that of the accessible surface.


Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr
Cell.: +33 (0)6.11.35.69.09



On 19/01/2011 16:46, Richard Edward Gillilan wrote:

Since several people have asked me for code, I now realize that I've 
contributed my bit to the confusion over names of various surface definitions.
To clear things up there is an excellent online article which covers the 
history of various molecular surface definitions that I highly recommend:

www.netsci.org/Science/Compchem/feature14e.html

Mainly there are two related surface definitions:

(a) solvent-accessible surface
(b) molecular surface = contact surface + reentrant surface

"solvent-excluded volume" has been defined using both of the above. Both (a) 
and (b) reduce to the same VDW surface when the solvent probe is zero.

There are also a number of smooth approximations to these definitions and 
others.


Richard Gillilan
MacCHESS


Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?

2011-01-14 Thread Nadir T. Mrabet

Good points Richard!

The ambiguity with surface definition starts with the assumption that 
atoms are (i) spheres and (ii) with fixed radii.


I am not sure Connolly was able to sell his original algorithm due to 
conflicts of interest with the Scripps, where it had been actually 
developped first.


How could I get the Varshney code?

Best regards,

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr
Cell.: +33 (0)6.11.35.69.09


On 13/01/2011 13:40, Richard Edward Gillilan wrote:

:

*Subject: **Re: [ccp4bb] What is the simplest method to analytically 
compute the Solvent-Accessible Surface Area of a given atom in a protein?*



My knowledge on this is probably quite out of date by now, but some 
years ago there was a lot of research on this topic because such 
surfaces are important in electrostatics and implicit solvation models 
(calculating surface area) as well as molecular graphics.


I think the most widely-used definition of a solvent-accessible 
surface is Lee-Richards surface in which a solvent-sized sphere is 
rolled along the surface of the protein. Surface is therefore 
rigorously defined as a piecewise collection of convex and concave 
patches of spheres and tori. It was Connolly who implemented (and 
sold) a practical algorithm for computing these surfaces. They were 
even known as Connolly surfaces and rendered as dots before modern 
computing hardware allowed for rendering surfaces. Several groups have 
developed high-efficiency versions of the calculation. Harold 
Scheraga's group, for example, has some FORTRAN code for this.  Fred 
Brook's virtual reality group also developed a high-effeciency 
parallel version (Varshney was the guy's name I think) in C.  There 
have been many approximations over the years I think ... but you asked 
about analytical models.


The these algorithms are non trivial. That's a understatement. And 
there is actually a mathematical ambiguity in the surface definition 
itself.


The Varshney code is freely available ... I received email permission 
from both Varshney and his thesis advisor to freely distribute the 
code. I even offered it to Warren Delano years ago when he was writing 
Pymol, but he refused to include it because he felt there still might 
be legal issues that would effect Pymol. So ... Pymol contains only a 
somewhat improvised an non-rigorous surface algorithm (last time I 
looked). Fine for graphics of course.


en.wikipedia.org/wiki/Accessible_surface_area 
<http://en.wikipedia.org/wiki/Accessible_surface_area>


Richard


On Jan 13, 2011, at 1:00 AM, Francois Berenger wrote:


Hello,

Does someone know some good articles on this particular topic?

I'd like to implement the thing myself, however if there is
a good software doing the job (with readable source code),
I might use and cite it.

Best regards,
Francois.





Re: [ccp4bb] Removing a tight binding ligand

2010-10-08 Thread Nadir T. Mrabet

 Hi,
It could help if you said what your ligand is.

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 08/10/2010 03:05, SIPPEL,KATHERINE H wrote:

Hi all,

I am working with a substrate binding protein. The protein scavenges 
its endogenous ligand out of the E. coli used for expression. I need 
to get this ligand out for both crystallographic and kinetic studies. 
I have tried denaturing in urea and refolding the protein with limited 
success. It refolds properly according to the CD spectra but it some 
how manages to hold on to trace amounts of ligand despite serial 
dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed 
by 50mM Tris. I also have a homolog that abjectly refuses to refold in 
either urea or guanidine, though it does turn the dialysis tubing into 
a lovely snow globe. There are alternative methods of performing the 
kinetics, but those will require destroying the protein which doesn't 
help on the crystallography front.


I was wondering if any of you out there had experience successfully 
removing very tightly bound ligands by an alternative method. I didn't 
see any mention on the subject in the archives. I had hoped you might 
be able to point me in the right direction.


Thanks for your time,

Katherine

Ph. D. candidate
Department of Biochemistry and Molecular Biology
College of Medicine
University of Florida



Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Nadir T. Mrabet

Thanks Jürgen.
Yes, you may show dissociation.
However, especially if you deal with assembly, then it might be 
difficult, if not impossible, to tell your exact subunit composition if 
you runBNP only in the first direction. Jacob mentions the possible 
occurrence of complex assemblies (AB, BB, ABB, AAB).


Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 19/05/2010 16:05, Jürgen Bosch wrote:

You don't necessarily need the second dimension.
BN-PAGE gel:
protein A alone| some proteins you know the size as reference|protein 
B alone| your mixture


You will be able to see in the mixture a) one or b) multiple bands, 
since the Coomassie is equally distributed and attached to your 
protein in a non-denaturing way it is directly proportional to the 
molecular mass of your protein complex.


I'm searching for the protocol which was I believe either from the 
Görg group or a group in Berlin, perhaps also Mann demonstrating 
dissociation of a multimeric complex in dependence of reducing agent.


If I find it I will post it tonight.

Jürgen

On May 19, 2010, at 10:01 AM, Nadir T. Mrabet wrote:


Maia speaks about native PAGE for which protein mobility (migration)
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a 2D
gel: Native in the first direction, then SDS-PAGE in the second one.
You actually need both data to infer stoechiometry and subunit 
composition.


Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet medecine.uhp-nancy.fr 
<http://medecine.uhp-nancy.fr>




On 19/05/2010 13:01, Jürgen Bosch wrote:

Not quite correct, look into Blue Native PAGE. There you can seperate
natively by mass.

Jürgen

..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On May 19, 2010, at 1:31, Maia Cherney <mailto:ch...@ualberta.ca>> wrote:



Dear Jacob, I offer  you my opinion.
Are you talking about electrophoresis? As far as I know it does not 
work

for the mass. The velocity of a protein depends on the charge at a
particular pH, the mass and shape of molecules etc. It's very difficult
to take all these things into consideration. Otherwise this would be a
very convenient method, much easier than the analytical centrifugation
or   gel-filtration that are usually used. However, electrophoresis 
does

not work for mass determination. Besides, complex formation hugely
depends on the protein concentration. If you dilute your mixture, your
complexes might dissociate. There is equilibrium constant between
different types of complexes.

Maia


Jacob Keller wrote:

Dear Crystallographers,

I am trying to optimize a native gel experiment of a two-protein
complex, running the smallest-detectable amount of protein component A
with varying amounts of component B.

 MWCharge MW/Charge
A   22 -5-4308
B   17-24 -702

This experiment is partly to determine stoichiometry, but also to
determine roughly the strength of the interaction.

B definitely runs much faster than A alone, as predicted, but I am
wondering what to expect with various oligomers. Should ABB run faster
or slower than AB? What about AABB? Theoretically, AA should certainly
run slower than A, and BB slower than B, simply because the
mass/charge ratio is the same, but the overall mass is greater. But
what happens when you have AAB, for example? There must be an equation
relating the mass/charge and mass (and perhaps gel percentage) to the
speed traveled in the gel--but what is the equation?

Thanks for your consideration,

Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu <mailto:j-kell...@northwestern.edu>
***






-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-

Re: [ccp4bb] Native Gel Theory and Practice

2010-05-19 Thread Nadir T. Mrabet
Maia speaks about native PAGE for which protein mobility (migration) 
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a 2D 
gel: Native in the first direction, then SDS-PAGE in the second one.

You actually need both data to infer stoechiometry and subunit composition.

Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 19/05/2010 13:01, Jürgen Bosch wrote:
Not quite correct, look into Blue Native PAGE. There you can seperate 
natively by mass.


Jürgen

..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On May 19, 2010, at 1:31, Maia Cherney  wrote:


Dear Jacob, I offer  you my opinion.
Are you talking about electrophoresis? As far as I know it does not work
for the mass. The velocity of a protein depends on the charge at a
particular pH, the mass and shape of molecules etc. It's very difficult
to take all these things into consideration. Otherwise this would be a
very convenient method, much easier than the analytical centrifugation
or   gel-filtration that are usually used. However, electrophoresis does
not work for mass determination. Besides, complex formation hugely
depends on the protein concentration. If you dilute your mixture, your
complexes might dissociate. There is equilibrium constant between
different types of complexes.

Maia


Jacob Keller wrote:

Dear Crystallographers,

I am trying to optimize a native gel experiment of a two-protein
complex, running the smallest-detectable amount of protein component A
with varying amounts of component B.

  MWCharge MW/Charge
A   22 -5-4308
B   17-24 -702

This experiment is partly to determine stoichiometry, but also to
determine roughly the strength of the interaction.

B definitely runs much faster than A alone, as predicted, but I am
wondering what to expect with various oligomers. Should ABB run faster
or slower than AB? What about AABB? Theoretically, AA should certainly
run slower than A, and BB slower than B, simply because the
mass/charge ratio is the same, but the overall mass is greater. But
what happens when you have AAB, for example? There must be an equation
relating the mass/charge and mass (and perhaps gel percentage) to the
speed traveled in the gel--but what is the equation?

Thanks for your consideration,

Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
***






Re: [ccp4bb] Query - inhibitor screening by fluorescence based assay.

2010-05-19 Thread Nadir T. Mrabet
You are measuring fluorescence changes which are likely to be due to 
compound binding by your enzyme.

In this case your blank must be your "compound" blank and no other.
Then you measure changes in fluorescence intensity and lambda max of 
emission as you add your enzyme.


I do not know you system.
Note however that binding (1) might be non specific and (2) need not 
correlate with enzyme inhibition.


Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr


On 19/05/2010 08:48, suresh mattegunta wrote:

Dear All,
Sorry for the non crystallography related question
We are performing a fluorescence based assay to screen for inhibitor 
compounds of our enzyme and ultimately crystallize the enzyme along 
with inhibitor.
We see that some of our compounds are autofluorescent and thus are 
effecting fluorescence. In such a case we make a compound blank (assay 
buffer+ compound) and subtract it from test (enzyme + compound) but 
still we see the values of test much higher compared to control (only 
enzyme). In this case can we bring down the values of compound balnk 
and test by a factor which brings the value of compound blank to the 
level of blank and compare the values of resultant test value with 
that of control after subtracting the respective blanks.
For example we have the following arbitrary fluorescent readings (AFU) 
for one compound:

Blank (70342) Control (163243) Compound balnk (1865830) Test (2020455)
Since compound blank / blank ie (1865830)/(70342) = 26.52 can we do a 
normalization of compound blank and test values by a factor 26.52, 
which brings teh value of compound blank to teh level of blank and get 
the values (1865830)/26.52 = 70355 and 2020455/26.52 = 76186 and say 
the compound is inhibitor by comparing this test value with the value 
of control after subtracting teh values of respective blank.

Thank you in advance,
Suresh


--
Suresh V Mattegunta MS
Senior Research Associate
Discovery Biology Division
Aptuit Laurus PVT LTD.
ICICI Knowledge Park
Hyderabad 500078 India


Re: [ccp4bb] Ion exchange protein lost

2010-05-12 Thread Nadir T. Mrabet

Human gcsf has a pI ~ 6.
As Ursula suggests you might be better off with anion exchange.
There are protocols that show you can both refold and purify onto such 
column type.


Nadir

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr
Cell.: +33 (0)6.11.35.69.09

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On 12/05/2010 19:17, Ursula Schulze-Gahmen wrote:
Like Juergen suggested, your protein is most likely stuck to the 
column, either because it never was really was refolded or because it 
doesn't like to be at pH 4.5. Do you really need such a low pH to have 
it bind to source 15S. Check the pI of the protein and perhaps some 
different pH buffer or try an anion exchange column if possible.



Ursula

On 5/11/10 7:53 PM, megha goyal wrote:

Hi all,
Our protein is gcsf. We solubilize the inclusion bodies in 8M urea 
and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% 
tween 20 pH 8.2. Then we perform concentration using proflux M12 
[just concentration and not diafiltration]. Adjust the pH of 
concentrate to 4.5 and load it to source 15S resin [strong cation 
exchange]. The problem is we do not recover our protein on performing 
IEX. HPLC and absorbance reading on concentrate show the presence of 
our protein. Buffer for loading is 25 mM na acetate pH 4.5 and 
elution is same buffer with 0.5 M NaCl. No protein is lost in flow 
thru and even 2M Nacl washing does not show our protein. . Only when 
we perform NaOH wash we do see some peak but could not analyse it as 
it is too alkaline and cant run on SDS PAGE or HPLC.


What could be the reason. Where do we lose our protein. Kindly shed 
some light on this on where shall I be going wrong.

thanks and regards,
meg


--
Ursula Schulze-Gahmen, PhD.
QB3, Tjian Lab
MCB, 16 Barker Hall #3204
University of California Berkeley
Berkeley, CA 94720-3204
Phone: (510) 642 8258
uschulze-gah...@lbl.gov

   


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

2010-05-11 Thread Nadir T. Mrabet

I would go for DTPA and acid pH.
Better than dialysis, I would use IEX and flush le bound protein with a 
buffer with as low a pH as possible with 10-100 mM DTPA, then wash with 
no DTPA, and elute le protein with clean (e.g. treated with Chelex) salt.


HTH,

Pr. Nadir T. Mrabet
Structural&  Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet  medecine.uhp-nancy.fr



On 11/05/2010 19:12, Javier Gonzalez wrote:
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 <mailto:rrowl...@colgate.edu>> wrote:


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 <mailto:rrowl...@colgate.edu>




Re: [ccp4bb] pdb-l: [mockeldri...@yahoo.com: Retraction of 12 Structures]

2009-12-11 Thread Nadir T. Mrabet

Kevin,

1hps and 1hos do no appear to include Murthy as an author.
Since I need good as well as (very) bad examples for my molecular 
modeling teaching,
I spent some time analyzing the structures which pdb codes you provided 
with MolProbity.
All score very bad: high B factors, lots of clashes, many Ramachandran 
outliers, unfavorable rotamers and bad bond angles.
The lesser bad structures in your list come (surprisingly?) as 1hos and 
1hps, but still they are far from optimal.

I have those data available in the case you need them.

I would strongly advise the use of MolProbity (at least) by both 
crystallographers and modelers, but also by curators at the rcsb..


Regards,

Nadir

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-954
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   




Kevin Karplus wrote:

Firas Khatib pointed out that several of the PDB files by Krishna
Murthy's group were identified as problematic in the RosettaHoles paper:

http://www.proteinscience.org/details/journalArticle/110975/RosettaHoles_Rapid_assessment_of_protein_core_packing_for_structure_prediction_r.html

I notice that the RosettaHoles paper also identifies 1BGX as a
problematic file, but that was not listed on the UAB site
http://main.uab.edu/Sites/reporter/articles/71570/

I also notice that most of the identified files have not yet been
marked as obsolete in PDB's "obsolete" file.  In fact, only 1BEF was
so marked when I just checked
ftp://ftp.wwpdb.org/pub/pdb/data/status/obsolete.dat

Question for the community:  should we remove ALL the PDB files from
Krishna Murthy's group as suspect?  I find the following in the
current PDB:

listed in article:  
1cmw 1df9 2qid 1g40 1g44 1l6l 2ou1 1rid 1y8e 2a01 2hr0

others probably from same author:   
1bgx 1ay1 1hef 1heg 1sbg 1hps 1hos

(Incidentally, variations in name abbreviations make it difficult to
be sure that I have found all of the files, or that all of the files
involve the same person.)

Has anyone examined 1bgx, 1ay1, 1hef, 1heg, 1sbg, 1hps, and 1hos to
see if there is any evidence of fraud on those files?  
I'm strongly tempted to remove them from my template libraries, unless

I hear from someone who was involved in the investigation that these
files were carefully examined and determined to be ok.  The fact that
1bgx was identified by RosettaHoles as suspect makes me wary of just
trusting that silence means they were cleared.


Kevin Karplus   karp...@soe.ucsc.eduhttp://www.soe.ucsc.edu/~karplus
Professor of Biomolecular Engineering, University of California, Santa Cruz
Graduate Director, Bioinformatics
(Senior member, IEEE)   (Board of Directors, ISCB)
Editorial Board, Bioinformatics (Oxford University Press)
life member (LAB, Adventure Cycling, American Youth Hostels)
Effective Cycling Instructor #218-ck (lapsed)
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Re: [ccp4bb] Van der Waals contacts

2009-07-15 Thread Nadir T. Mrabet

Totally agree with what you write.
Yet, I was only answering the question asked (that dealt mostly with
"van Der Waals contacts and effective contacts").
At a time of crisis (DeltaG = only a few kcal/mol), I would say 0.6 kcal/mol
would be a little more than negligible. Question of balance.

Nadir

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-954
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   




Bernhard Rupp wrote:

It might be worthwhile to consider the energy column in the pdf:
At RT we have about 0.6 kcal/mol thermal energy, so a
*single attractive* vdW interaction has little impact - 
it is generally the sum of many of those contributing to
notable and important attractive forces. 
For a *single repulsive* (steep branch) vdW this is
quite different - your quickly get energetically significant 
repulsions even for one bad contact. So the vdW repulsion are both 
useful (nearly independent) stereochemical restraints and a good 
indicator for improbable conformations (see molprobity clash score, 
rama, etc).


BR

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nadir
T. Mrabet
Sent: Wednesday, July 15, 2009 7:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Van der Waals contacts

This issue is far from being trivial.

To address the question, I recommend looking at this simple example
(attached pdf file) which shows that even when the atom distance is twice
the vdw diameter, there is still attraction between atoms (unified type,
with implicit H atoms).

Some, but not all, consider that water screens out interactions, that is,
the vdw interaction is attractive provided that R < Rm + 2 x R(H2O).
But then, one may also need to consider the special case of water-mediated
H-bonds, where the heavy atoms being considered are polar.

HTH,

Nadir


Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: nadir.mra...@medecine.uhp-nancy.fr
Cell.: +33 (0)6.11.35.69.09


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Clayton, Gina Martyn wrote:
  

Hi CCP4ers

Perhaps I am hashing over old news...but

We are having a discussion about Van Der Waals contacts and effective 
contacts i.e. the "real distance" of a VDW bump between say a CH and a 
CH group which sometimes is described as between a C and a C as i.e.

2x 1.6A and ending about 4A but not including hydrogen.

Some programs list contacts, to say a ligand, as far as 6A apart and 
some of the simulation programs use that distance too for contacts for 
protein protein interactions.


Does anyone know of a good paper that discusses the effective distance 
or has a comment on where a VDW force may begin and end or it's 
effective distance - though some say it never truly ends just 
approaches zero...



G



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Re: [ccp4bb] structure <-> function

2009-06-22 Thread Nadir T. Mrabet

Ok, now we can perhaps debate of another problem.
With a multiple choice question that has more than one acceptably good 
answer,

is it "convergent", or rather "independent", evolution?

This multiple choice question is open for discussion.

Greetings,

Nadir

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-954
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
  




Justin Lecher wrote:

Many thanks to everyone who replied. Once again a proof of this community!

I am sure I will find what I am looking for in the cited publications.

Thanks,
justin



  


Re: [ccp4bb] Glycerol as metal chelator?

2009-05-14 Thread Nadir T. Mrabet

I wouldn't use a pH of 9 when dealing with metal cations in solution.
By and large, metal hydroxides precipitate at alkaline pH.
HTH

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-954
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   




Guenter Fritz wrote:

Ho,
interesting case.
Glycerol actually forms weak complexes with metal ions:
Journal of Inorganic Biochemistry,60 299-302 (1995) and some 
references therein.
But to be true, I am surprised that glycerol at concentrations of 
10-15% (?)  as used in cryo conditions can compete with a metal ion 
binding site in a protein. I remember I myself did titrations with 
Zn2+ in the presence/absence of 5% glycerol and did not observe any 
effect on the binding. There might be another effect (?) although I 
have no idea what it might be.



 I am working with a metalloprotein that binds cobalt and iron. I
was surprised that the solved structures showed the crystals
cryoprotected with glycerol are metal free while crystals
cryoprotected with ethylene glycol had the metals present. Both
cryoprotectant solutions contained metal in the 10 mM range and are
buffered at pH 9. I assume glycerol must be a weak chelator otherwise
it wouldn't be so ubiquitous in protein biochemistry. Has anyone else
experienced this before with glycerol?


Ho
UC Berkeley
  







Re: [ccp4bb] Van der Waals Interaction Distances

2009-02-20 Thread Nadir T. Mrabet

Hi,

This is one table (below) I use to convince my students that Evdw is 
still negative when R/Rm = 2, where Rm = Ri + Rj.
To take into account water screening effect, however, one should 
consider vdw attraction only while R < Rm + 2 x Rw,

where Rw is the radius of water.

hth,

Nadir

**

**



**



**



**

*1.00*



*1.000*



*1.000*



*-0.130*

*1.05*



*0.936*



*1.069*



*-0.122*

*1.10*



*0.810*



*1.234*



*-0.105*

*1.15*



*0.678*



*1.475*



*-0.088*

*1.20*



*0.558*



*1.793*



*-0.072*

*1.25*



*0.456*



*2.195*



*-0.059*

*1.30*



*0.371*



*2.692*



*-0.048*

*1.35*



*0.303*



*3.299*



*-0.039*

*1.40*



*0.248*



*4.033*



*-0.032*

*1.45*



*0.204*



*4.911*



*-0.026*

*1.50*



*0.168*



*5.957*



*-0.022*

*1.55*



*0.139*



*7.193*



*-0.018*

*1.60*



*0.116*



*8.646*



*-0.015*

*1.65*



*0.097*



*10.346*



*-0.013*

*1.70*



*0.081*



*12.324*



*-0.011*

*1.75*



*0.068*



*14.616*



*-0.009*

*1.80*



*0.058*



*17.260*



*-0.008*

*1.85*



*0.049*



*20.298*



*-0.006*

*1.90*



*0.042*



*23.776*



*-0.005*

*1.95*



*0.036*



*27.742*



*-0.005*

*2.00*



*0.031*



*32.252*



*-0.004*



--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   




Jim Fairman wrote:

Fellow CCP4 Board Members,

What is the general consensus of the structural biology community for 
a range of distances that would be considered a Van der Waals 
contact/interaction (eg: hydrogen bonds are usually considered to be 
2.5-3.5 angstroms not including the hydrogen atoms)?


Cheers, Jim 


--
Jim Fairman
Graduate Research Assistant
Department of Biochemistry, Cellular, and Molecular Biology (BCMB)
University of Tennessee -- Knoxville
216-368-3337 jfair...@utk.edu <mailto:jfair...@utk.edu> 
james.fair...@case.edu <mailto:james.fair...@case.edu>


Re: [ccp4bb] PDB protein strucutrues as screen saver

2009-02-16 Thread Nadir T. Mrabet

Hi,
You may want to have a look at 
http://www.luminorum.com/html/luminorum_ltd___extras.html.

hth
Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   



Jayashankar wrote:

Dear Scientists,

It may be too much...

But as a biophysics student I would like to appreciate and feel happy 
to have pdb
structures as my computers screen savers than to have some funny and 
fancy stuffs.

And it may help me as a motivator to solve my own structures in future

I want to ask is there any existing script that grep strucutres one by 
one with one line definition of that structure.





S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


Re: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space

2009-02-10 Thread Nadir T. Mrabet

Artem,

Artem Evdokimov wrote:

Please note that osmotic shock extraction typically employs EDTA which is
obviously bad for IMAC. 
  

This is not entirely correct.
I have used extracts with 5 mM EDTA for IMAC in the past.
If your IMAC column volume is large enough, only the top 1-2 mm will be
depleted of Me2+ (easily seen with Cu2+).
Moreover, it is always possible to add some Me2+ to your extract prior 
to IMAC.

All this was published long ago (Biochemistry. 31: 2690-2702, 1992).

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
  


Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Nadir T. Mrabet

Two things to be mentioned.
* IDA columns bear an overall negative charge. I expect this behavior 
holds true with NTA gels. Hence salt, (>= 0.5 M NaCl) must be present in 
your adsoprtion buffer to quench possible repulsive electrostatic 
interactions.
* You are dealing with protein adsoption by coordination bond formation 
to a metal-chelate. Coordination bond lentghs decrease (and binding 
improves) as ionic strength increases, so a 1-2 M salt concentration in 
you buffer may turn out to be appropriate.


HTH,

Nadir Mrabet

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: nadir.mra...@medecine.uhp-nancy.fr
   



Fred wrote:

Hi everyone,
Thanks for answer my question. Just to add some more notes regarding 
to my expression system. The insert-vector (pET28) has been sequenced 
and the his-tag is N-terminal. The anti his-tag WB is positive and the 
binding buffer's pH is 8.2 (double-checked).
I had already experienced the same problem before, which I solved just 
increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no 
binding at all.
I'm currently running a SDS-PAGE with samples eluted from Talon and 
let you know the results.

All the Best,
Fred 



--- Fred // schrieb am *Di, 27.1.2009:
*

*Von: Fred 
Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 27. Januar 2009, 22:00

*

*Hi ccp4 list,
I am trying to purify a his-tag protein by metal affinity 
chromatography. The
protein was expressed in inclusion bodies and its his-tag doesn't 
bind the
Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). 
Playing with

NaCl and detergents didn't help much.
Any help is appreciated.
Fred   *







Re: [ccp4bb] Definition of salt bridge

2008-10-16 Thread Nadir T. Mrabet

The problem lies with your definition of "significant".
If it is non null, then any interaction is significant (dual-pan balance 
concept).
Coulomb's energy is a function of 1/r^2, therefore at 8 Angs, it is 
still 15% of Emax.

Even H-bonds are sometimes considered relevant up to 5 Angs.

Nadir Mrabet

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   




Ibrahim Moustafa wrote:

Yes, it is electrostatic interaction. But when searching for a salt-bridge
in a protein structure it won't be considered a significant non-bonded
interactions at 8 A distance. Also, the electrostatic interaction extends
beyond 8 A. For a significant interaction the distance need to be < 8A.

  Ibrahim


On 10/16/08 12:10 PM, "Nadir T. Mrabet" <[EMAIL PROTECTED]>
wrote:

  

--

Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax:   +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED]



Hi,

Salt bridges (or ion pairs) can be long-range (up to 7-8 Ang). They obey
Coulomb's law.
In contrast, H-bonds are short-range and are further anisotropic.

For those with general interest in electrostatics, I suggest to go back
to the
1978 paper of Max Perutz:
Electrostatic Effects in Proteins
Science (1978) 201 (4362), 1187-1191.

Nadir Mrabet

Jayashankar wrote:


Dear Fransico,

*Salt bridges are close range electrostatic interaction which depend
on conformer population.

*S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Thu, Oct 16, 2008 at 8:21 AM, Chavas Leo <[EMAIL PROTECTED]
<mailto:[EMAIL PROTECTED]>> wrote:

Dear Francisco --

On 15 Oct 2008, at 17:05, Francisco J. Enguita wrote:
  

how

can you define a salt-bridge within a protein structure ?



According to Wikipedia:
a salt bridge in proteins is "a relatively weak ionic bond between
positively and negatively charged side-chains of proteins."

Now, at far as I understand (based on "Structure and Mechanism in
Protein Science - Alan Fersht), you have a salt bridge when two
groups are making an hydrogen bond that is favored by
electrostatic interaction, electrostatic energies being weak in
water. To quote the author of the book, let say you have the
following equilibrium:

E-NH3+  ---  OH2   +   OH2  ---  -O2C-S  <==>  E-NH3+
 ---  -O2C-S   +   H2O  ---  H2O

The right-hand side equation would be more "favorable", as the
electrostatic interaction will be more stable than in the
left-hand side where both ions would be in contact with water
molecules. 


HTH

Kind regards.

-- Leo --

Chavas Leonard, Ph.D. @ home
Research Associate
Marie Curie Actions Fellow

Faculty of Life Sciences
The University of Manchester
The Michael Smith Building
Oxford Road
Manchester Lancashire
M13 9PT

Tel: +44(0)161-275-1586
e-mail: [EMAIL PROTECTED]
<mailto:[EMAIL PROTECTED]>
http://personalpages.manchester.ac.uk/staff/leonard.chavas/



  



  


Re: [ccp4bb] Definition of salt bridge

2008-10-16 Thread Nadir T. Mrabet

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   


Hi,

Salt bridges (or ion pairs) can be long-range (up to 7-8 Ang). They obey 
Coulomb's law.

In contrast, H-bonds are short-range and are further anisotropic.

For those with general interest in electrostatics, I suggest to go back 
to the

1978 paper of Max Perutz:
Electrostatic Effects in Proteins
Science (1978) 201 (4362), 1187-1191.

Nadir Mrabet

Jayashankar wrote:

Dear Fransico,

*Salt bridges are close range electrostatic interaction which depend 
on conformer population.


*S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Thu, Oct 16, 2008 at 8:21 AM, Chavas Leo <[EMAIL PROTECTED] 
<mailto:[EMAIL PROTECTED]>> wrote:


Dear Francisco --

On 15 Oct 2008, at 17:05, Francisco J. Enguita wrote:


how

can you define a salt-bridge within a protein structure ?



According to Wikipedia:
a salt bridge in proteins is "a relatively weak ionic bond between
positively and negatively charged side-chains of proteins."

Now, at far as I understand (based on "Structure and Mechanism in
Protein Science - Alan Fersht), you have a salt bridge when two
groups are making an hydrogen bond that is favored by
electrostatic interaction, electrostatic energies being weak in
water. To quote the author of the book, let say you have the
following equilibrium:

E-NH3+  ---  OH2   +   OH2  ---  -O2C-S  <==>  E-NH3+
 ---  -O2C-S   +   H2O  ---  H2O

The right-hand side equation would be more "favorable", as the
electrostatic interaction will be more stable than in the
left-hand side where both ions would be in contact with water
molecules. 


HTH

Kind regards.

-- Leo --

Chavas Leonard, Ph.D. @ home
Research Associate
Marie Curie Actions Fellow

Faculty of Life Sciences
The University of Manchester
The Michael Smith Building
Oxford Road
Manchester Lancashire
M13 9PT

Tel: +44(0)161-275-1586
e-mail: [EMAIL PROTECTED]
<mailto:[EMAIL PROTECTED]>
http://personalpages.manchester.ac.uk/staff/leonard.chavas/





Re: [ccp4bb] off-topic: Enzymology textbook recommendations?

2008-09-12 Thread Nadir T. Mrabet

Alan Fersht: Enzyme structure and mechanism.
Definitely!
Nadir Mrabet

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   



Donnie Berkholz wrote:

On 13:37 Thu 11 Sep , Tim Fenn wrote:
  

On Thu, 11 Sep 2008 13:24:12 -0700 William Scott
<[EMAIL PROTECTED]> wrote:


I just found out that in a couple of weeks I am going to be teaching
a graduate student-level enzymology course.

Can anyone recommend a good text, and possibly good websites
authored by people who are predisposed to consider plagiarism the
highest form of complement?
  

A few classics I wouldn't be caught without:

"Catalysis in Chemistry and Enzymology" William P. Jencks
"Enzymatic Reaction Mechanisms" Christopher Walsh



Our resident enzymologist (who just retired) recently picked up a copy 
of a current book that claims to update Jencks and Walsh, since they 
were both published before 1980. It's by Frey & Hegeman: 
http://www.oup.com/us/catalog/he/subject/Chemistry/Biochemistry/ProteinsandEnzymes/?view=usa&ci=9780195122589


If you try it out, I'd love to hear how it goes.

  


Re: [ccp4bb] Na Acetate Buffer

2008-07-23 Thread Nadir T. Mrabet
Michael's comments are correct and follow indeed appropriate chemistry 
theory.
In practice however, as said earlier, it is less likely to read a 
correct pH value with a pH-meter (unless extensive care and adjustments 
are taken beforehand) than to predict amounts of acid and base to use 
reliably based on the HH equation.


I take this opportunity to correct my mistyping in my previous mail: "In 
the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - [NaOH])"
should be rewritten into "In the second case, the HH is written 4.5 = 
4.76 + log([NaOH]/(25 - [NaOH]))".


Greetings,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   




R.M. Garavito wrote:


Buffer making is very much an empirical process, but there is a 
comment that needs to be made about the use of the H-H equation and 
pKa values. I have to teach our department's biochemistry laboratory, 
and I sadly would have to take off points from all the discussions as 
the H-H equation and pKa won't give the right answer as pKa is for an 
ideal (i.e., infinitely dilute) solution. A 25 mM Na acetate solution 
is not dilute. You need to use the pKa' which sadly changes as the 
concentration of the buffer increases or decreases: while the pKa of 
acetic acid is 4.76 (@25˚C), at 100mM, the pKa' is 4.60. The National 
Bureau of Standards (now NIST) has a detailed list of standard buffer 
recipes and pKa' values for most of the common buffers (e.g., see 
Bates, J. Natl. Bur. Stand. 66A, 179, 1962).


That said, Nadir's method is a fine way to make a buffer of a known pH 
(using a well calibrated pH meter) at a known temperature, and it will 
allow you to make a buffer with the same pH value almost every time 
(depending on how your room temperature changes throughout the year). 
Being able to consistently and reliably repeat the buffer formulation 
is the most important point.


Cheers,

Michael

//

/R. Michael Garavito, Ph.D./

/Professor of Biochemistry & Molecular Biology/

/513 Biochemistry Bldg. /

/Michigan State University /

/East Lansing, MI 48824-1319/

/Office:// //(517) 355-9724 Lab: (517) 353-9125/

/FAX: (517) 353-9334 Email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>/

/************/



On Jul 22, 2008, at 11:20 AM, Nadir T. Mrabet wrote:

I bet it is more difficult to adjust a pH-meter than to use the 
Henderson-Hasselbalch equation
and still get the expected pH with a pretty good accuracy especially 
if your work near the pKa.


There are actually two ways to prepare this 25 mM buffer, pH 4.5.

The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so 
don't worry too much about this).
Reference is "Buffers for pH and Metal Ion Control", Perrin & 
Dempsey, Chapman & Hall, NY, ISBN 0 412 21890 9.


High-grade glacial acetic acid (99-100%) is 18 N.
Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final). Keep 
is a dark, tightly closed bottle.


Make a stock solution of 250 mM sodium acetate (if you use FW, not 
MW, to calculate mass to use, then no worry about anhydrous or not 
since water is also taken into account if present)


or

make a stock solution of 5N NaOH. Keep is a dark, tightly closed bottle.

Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log 
([A-]/[AH]).


In the first case, you write it : 4.5 = 4.76 + log ([sodium 
acetate]/[acetic acid])

Second equation is [sodium acetate] + [acetic acid] = 25 mM
which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134 mM.
For 1.0 L buffer, mix adequate volumes of stock solutions of sodium 
acetate and acetic acid and complete with water (add acid after un 
first fill with water to ~ 800 mL).


In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - 
[NaOH]),
which gives [NaOH] = 8.886 mM (same result as above for sodium 
acetate which was then the base).


The added advantage of using HH and stock solutions is that even if 
your pH is not exactly 4.5, say 4.55, if you make a new buffer the 
next day or even the next month,
your buffer will have the same pH value. I don't expect you can ever 
achieve such a repeatability using a pH-meter.


HTH,

Nadir Mrabet

--

Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED] 
<mailto:[EMAIL PROTECTED]>





William G. Scott wrote:
So what, then, will be the concentration of the acetate ion in your 
stock so

Re: [ccp4bb] Na Acetate Buffer

2008-07-22 Thread Nadir T. Mrabet
I bet it is more difficult to adjust a pH-meter than to use the 
Henderson-Hasselbalch equation
and still get the expected pH with a pretty good accuracy especially if 
your work near the pKa.


There are actually two ways to prepare this 25 mM buffer, pH 4.5.

The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so don't 
worry too much about this).
Reference is "Buffers for pH and Metal Ion Control", Perrin & Dempsey, 
Chapman & Hall, NY, ISBN 0 412 21890 9.


High-grade glacial acetic acid (99-100%) is 18 N.
Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final). Keep is a 
dark, tightly closed bottle.


Make a stock solution of 250 mM sodium acetate (if you use FW, not MW, 
to calculate mass to use, then no worry about anhydrous or not since 
water is also taken into account if present)


or

make a stock solution of 5N NaOH. Keep is a dark, tightly closed bottle.

Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log 
([A-]/[AH]).


In the first case, you write it : 4.5 = 4.76 + log ([sodium 
acetate]/[acetic acid])

Second equation is [sodium acetate] + [acetic acid] = 25 mM
which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134 mM.
For 1.0 L buffer, mix adequate volumes of stock solutions of sodium 
acetate and acetic acid and complete with water (add acid after un first 
fill with water to ~ 800 mL).


In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - 
[NaOH]),
which gives [NaOH] = 8.886 mM (same result as above for sodium acetate 
which was then the base).


The added advantage of using HH and stock solutions is that even if your 
pH is not exactly 4.5, say 4.55, if you make a new buffer the next day 
or even the next month,
your buffer will have the same pH value. I don't expect you can ever 
achieve such a repeatability using a pH-meter.


HTH,

Nadir Mrabet

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]




William G. Scott wrote:
So what, then, will be the concentration of the acetate ion in your 
stock solution when you have finished?


(Disclaimer:  I get to teach this stuff periodically in remedial 
chemistry as a punishment for deployment of excessive sarcasm during 
faculty meetings.)


On Jul 22, 2008, at 6:10 AM, Santosh wrote:


Hi,
Make a  1M Na-Acetate do not make up to the 1 Ltr volume. Leave some 
extra
volume and now start adding Acetic acid till you get pH 4.5 (Glacial 
Acetic

Acid).
Now make up the volume to 1ltr or how much ever you are deciding to 
make the

50X stock solution.
Best,
Santosh

On Mon, Jul 21, 2008 at 11:20 PM, William G. Scott <
[EMAIL PROTECTED]> wrote:


This is a job for the trusty Henderson-Hasselbalch equation:

http://en.wikipedia.org/wiki/Henderson-Hasselbalch_equation



On Jul 21, 2008, at 8:12 PM, Meg wrote:

Dear All,


I want to prepare 25 mM sodium acetate buffer pH 4.5. can anyone 
give the
exact composition of how to prepare it. we prepare it using sodium 
acetate
and acetic acid combination. i am not able to arrive at the 
calculatation

correctly, so if anyone can  explain me with the above buffer how to
calculate. and what sodium acetate [Anhydrous / trihydrate] and acetic
acid
[glacial/ plain] to use.

thanks n regards

Meg goyal,
M.SC Biotechnology [Research]
Institute of science,
Fort
Mumbai, INDIA








Re: [ccp4bb] Imidazole's ability to chelate metal ions

2008-07-18 Thread Nadir T. Mrabet

Well, I happen to lecture on this...
The bible is "Data for biochemical research", Dawson et al., Oxford 
University Press.

Should be available in most biochemistry labs, if not in the library.

Cheers,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   


Jacob Keller wrote:
Could those who responded with numbers for affinities of imidazole for 
metal ions please divulge their sources? It is not that I doubt their 
veracities, but it would be a nice reference to have on hand.


For those wondering about why I was asking about imidazole's affinity 
for metal ions, I was wondering whether the presence of imidazole 
would affect a metal-ion-dependent reaction. With, for example, 200 mM 
imidazole and 10 mM Ca++ or Mg++, what would be the amount of free 
metal? This can of course be calculated from imidazole's binding 
constant for these ions, which is another reason I ask for the sources 
of the numbers quoted in a couple of the responses.


Thanks for all of the helpful responses so far,

Jacob Keller


***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

- Original Message - From: "Nadir T. Mrabet" 
<[EMAIL PROTECTED]>

To: "Jacob Keller" <[EMAIL PROTECTED]>
Cc: 
Sent: Friday, July 18, 2008 8:55 AM
Subject: Re: [ccp4bb] Imidazole's ability to chelate metal ions


Imidazole can indeed complex (monodentate) metal ions but not chelate 
them (bidendate, at least).
However, the stability constant, K, of such complexes is rather low, 
eg log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu.
In comparison, metal chelates are formed with EDTA, for which log K = 
10.6 for Mg, 14.2 for Fe and 18.8 for Cu.

So the difference amounts to several orders of magnitude.

It should also be pointed out that the competitive effect of 
imidazole in IMAC does not involve binding to free metal ions,
but instead coordination to immobilized metal chelates, eg 
Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator).


In any situation where one assays a protein whose activity and/or 
stability and/or else is/are metal dependent, one should
rather use buffers (see below) that do not interfere (eg Good's 
buffers).


I suspect the imidazole in your case is either a buffer (pKa 7.0) or 
else results from competitive elution from an IMAC column.

What should be done depends on your exact conditions.

hth,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]



Jacob Keller wrote:

Dear Crystallographers,
 Does anybody happen to know whether imidazole is able to chelate 
metal ions in solution? It seems reasonable that since it can 
compete for binding to IMAC resins, it should have some affinity for 
at least Ni++ and Co++, but what about metal ions like Ca++ and 
Mg++? I assume that the affinity is weak, but at the concentrations 
at which we are wont to use it in our elutions (~100-500 mM), does 
it not seem likely that other metal ions are being competed away 
from our proteins as well?

 Jacob Keller
 ***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
***







Re: [ccp4bb] Imidazole's ability to chelate metal ions

2008-07-18 Thread Nadir T. Mrabet
Imidazole can indeed complex (monodentate) metal ions but not chelate 
them (bidendate, at least).
However, the stability constant, K, of such complexes is rather low, eg 
log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu.
In comparison, metal chelates are formed with EDTA, for which log K = 
10.6 for Mg, 14.2 for Fe and 18.8 for Cu.

So the difference amounts to several orders of magnitude.

It should also be pointed out that the competitive effect of imidazole 
in IMAC does not involve binding to free metal ions,
but instead coordination to immobilized metal chelates, eg 
Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator).


In any situation where one assays a protein whose activity and/or 
stability and/or else is/are metal dependent, one should

rather use buffers (see below) that do not interfere (eg Good's buffers).

I suspect the imidazole in your case is either a buffer (pKa 7.0) or 
else results from competitive elution from an IMAC column.

What should be done depends on your exact conditions.

hth,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   




Jacob Keller wrote:

Dear Crystallographers,
 
Does anybody happen to know whether imidazole is able to chelate metal 
ions in solution? It seems reasonable that since it can compete for 
binding to IMAC resins, it should have some affinity for at least Ni++ 
and Co++, but what about metal ions like Ca++ and Mg++? I assume that 
the affinity is weak, but at the concentrations at which we are wont 
to use it in our elutions (~100-500 mM), does it not seem likely that 
other metal ions are being competed away from our proteins as well?
 
Jacob Keller
 
 
***

Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
***


Re: [ccp4bb] pH gradient in Mono Q

2008-06-25 Thread Nadir T. Mrabet

John,

You can adjust your ionic strength not only with NaCl/KCl/etc but also 
by increasing the concentration of the components
in your buffer mixture. If you do it right, I can assure you can get a 
linear pH gradient.
And, as mentioned earlier by Tom, you can use both pH and salt gradients 
to refine your separation, even batchwise (no linear

gradient required... if it works).

The problem working with pH gradients is re-equilibrating the resin, 
since what you are actually doing is titrate a supposedly
high-capacity buffer with another high-capacity buffer. Therefore, 
patience is required and sufficient volumes of buffer A.


Again, I ask the question: Did Matthew ever mentioned before he intended 
to perform a CHROMATOFOCUSING

experiment?

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   




John A. Newitt wrote:

At 1:50 PM -0400 6/24/08, R.M. Garavito wrote:


Matthew,

You're not going to ruin your column, but you won't get great 
performance either.  Elution by pH change is a very common method, 
but getting a really linear pH gradient is very hard.  The Mono Q 
matrix is a strong anion exchanger, meaning that it is insensitive to 
pH changes, i.e., you can't titrate it smoothly with acid or base.  
DEAE resins, which are weak anion exchangers, have a nice pH 
titration curve and lend themselves better to elution by pH change. 
This is the reason chromatofocusing is not a commonly used method, 
and its expensive.


There is a company the sells a proprietary buffer system and gradient 
programming calculator to create a stable pH gradient for separation 
on a MonoQ column or other strong ion exchanger.


<http://www.cryobiophysica.com/>

My problem with pH gradient techniques is that they don't work very 
well unless your protein is happy in low ionic strength buffers, which 
is almost never the case with my projects. This company now claims 
that it can create the pH gradient with NaCl present, but I haven't 
tried this yet.


- John


Re: [ccp4bb] pH gradient in Mono Q

2008-06-24 Thread Nadir T. Mrabet

Michael,

Well, why do you need to titrate the exchanger rather then the proteins 
themselves?
MonoQ is a lot simpler to adjust pHwise, as with DEAE you actually 
titrate both the matrix and the proteins.
Recommended buffers to use are Goods' (pKa from 8 to 6.15 at 20 °C) + 
acetic acid (pKa 4.76).
An equimolar (50mM) mixture of these with Buffer A titrated to 8.0 and 
Buffer titrated to 4.0 has been
shown (in  my hands) to yield a very linear gradient (must not be too 
steep, though).


Matthew's question does not seem to concern chromatofocusing.

Hth,

Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   



R.M. Garavito wrote:

Matthew,

You're not going to ruin your column, but you won't get great 
performance either.  Elution by pH change is a very common method, but 
getting a really linear pH gradient is very hard.  The Mono Q matrix 
is a strong anion exchanger, meaning that it is insensitive to pH 
changes, i.e., you can't titrate it smoothly with acid or base.  DEAE 
resins, which are weak anion exchangers, have a nice pH titration 
curve and lend themselves better to elution by pH change.  This is the 
reason chromatofocusing is not a commonly used method, and its 
expensive.  

Andreas has pointed you in the general direction for chromatofocusing, 
but there is a "poor man's" way to do it.  We use this method a lot, 
and the key is using a weak ion exchanger (like DEAE or CM) and a mix 
of buffers with pKas that span the titration range you want to 
exploit.  Remember, you actually want to titrate the resin with the 
buffer: as the pH shifts away from the pKa of one buffer component, it 
moves into the buffering range of the other.  If you do it correctly, 
you get a nice, flatter titration curve from the resin, which spreads 
out the release of the proteins.  We have used a mixture of Tris and 
Bis-Tris-Propane with a HiTrap-DEAE or Sepharose-DEAE FF columns.


Hope this helps,

Michael

//

/R. Michael Garavito, Ph.D./

/Professor of Biochemistry & Molecular Biology/

/513 Biochemistry Bldg.   /

/Michigan State University  /

/East Lansing, MI 48824-1319/

/Office://  //(517) 355-9724 Lab:  (517) 353-9125/

/FAX:  (517) 353-9334Email:  [EMAIL PROTECTED] 
<mailto:[EMAIL PROTECTED]>/


//



On Jun 24, 2008, at 12:53 PM, Matthew Chu wrote:


Dear All,

Sorry for off-topic question. Does anyone have any experience in 
purifying protein using pH gradient in Mono Q column?


I have been googling for a whole day, only one paper was found to 
mention performing pH gradient in Mono Q, but in a mixture of amine 
buffering species, which is a bit too complicated (J. Chromatogr. A 
1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give 
a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH 
gradient in Mono Q as I don't want to ruin my Mono Q column...


Any suggestions are welcome. Thanks in advance!

Kind regards,
Matt

 


Matthew LH Chu
PhD Student
School of Pharmacy and Pharmaceutical Sciences
University of Manchester





Re: [ccp4bb] 3D model building without CRT monitor

2008-06-02 Thread Nadir T. Mrabet

Hi,
I bought myself a brand-new Viewsonic P227f crt less than a year ago (in 
France).

So I suspected this item to be still available. But no longer!.
I however found something that might still be of interest to you 
(http://www.viewsonic.com/products/crtmonitors/graphicseries/g90fB/).

Seems to be the one and only crt from Viewsonic (but only 19").
Cheers,
Nadir

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   Cell.: +33 (0)6.11.35.69.09



Krojer,Tobias wrote:

Dear all,
 
recently some of our CRT monitors broke down and we realized that 
these monitors are no longer produced. However, we would still like to 
continue model building in 3D which is apparently not possible with 
current TFT displays. Beside the possibility of getting old CRT 
monitors at ebay, I was wondering how other groups solved this problem.
 
Thank you very much for suggestions!

best wishes,
Tobias
 
 
 
 
Tobias Krojer, PhD

IMP (Research Institute of Molecular Pathology)
Dr. Bohr-Gasse 7
1030 Vienna
Austria
Tel.: +43-1-797303358
Fax.: +43-1-7987153


Re: [ccp4bb] crashing-out protein eluted from Nickel column

2008-02-18 Thread Nadir T. Mrabet

Hi,

One has to bear in mind that adsorption at high pH (above 6.5 is already 
high for Imac; pKa of exposed His is 6.2) leads to stronger interactions 
which, in turn, require stronger eluting conditions, e.g. > 200 mM 
imidazole (pKa ~ 7.0).
I suggest lowering the pH of the adsortion buffer, e.g. 6.5 or even 6.0 
(try and see) so that you dont need to go beyond 100 mM
imidazole in your elution buffer (as mentioned earlier, you must ajust 
the pH of the latter as required with extra HCl).


Best,

Nadir Mrabet

--

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   Nancy University, School of Medicine
   9, Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]




Ronnie Berntsson wrote:

Hi,

In addition to the tips already suggested, you could also try to elute 
your protein with histidine.. I've got a protein that crashes out with 
imidazole as well, and I have succesfully used 200mM histidine for 
elution (using either KPi or MES buffer in my case).


Cheers,
Ronnie

On Feb 15, 2008, at 5:47 PM, Juliana Barbosa Coitinho wrote:



Dear all, 
I am with the same problem of Jacob. 
My protein is precipitating, especially when I 'freeze it'. I am 
using a His Trap HP column coupled with a desalting column. Already 
tried to elute with gradient of imidazole (that allowed an elution 
with less amount of imidazole), but even then, the protein still 
crashes-out.
I am trying to use the protein immediately after purified it, but 
this is not always possible and I am losing much protein with that. 
I will try to use the tips that have already been said. 
But if someone can help me more, I will thank!!!


Thanks a lot!

Juliana


Abra sua conta no Yahoo! Mail 
<http://br.rd.yahoo.com/mail/taglines/mail/*http://br..mail.yahoo.com/>, 
o único sem limite de espaço para armazenamento!




Ronnie Berntsson

--
Ph.D. Student
Department of Biochemistry
Groningen Biomolecular Sciences and Biotechnology Institute
& Zernike Institute for Advanced Materials
University of Groningen
Nijenborgh 4, 9747 AG
Groningen, The Netherlands

telephone: +31 50 363 4195
telefax: +31 50 363 4165
e-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
homepage: http://www.rug.nl/gbb/research/researchgroups/enzymology/index





Re: [ccp4bb] Primary source for detergent properties

2007-10-23 Thread Nadir T. Mrabet

Hi,

The only published compendium on detergents still available (to my 
knowledge) is http://www.merckbiosciences.co.uk/docs/docs/LIT/CB0068_M.pdf.

No phase diagrams though.

Regards,

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   UHP - Nancy 1, School of Medicine
   Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   Cell.: +33 (0)6.11.35.69.09



R.M. Garavito wrote:

Jacob,

Actually, there is no real definitive compendium of detergents and 
their properties (solubility, CMC, aggregation number, etc.).  Because 
of their importance to industry, commercial compendia, as Anatrace's 
catalog, are often the most complete.  However, most commercial 
compendia are not focused on the detergents we are interested in; they 
want to make dishes cleaner while protecting your hands (i.e., SDS is 
good).  One of the best sources for detergent information of all sorts 
is Surfactants and Interfacial Phenomena by M. J. Rosen, but is is 
dated and out of print, I believe.  

Academic sources are few and often have circular citations.  I can 
name several cases in the more popular reviews that cannot be traced 
back to a original reference.  All the real biochemists and chemists 
who really brought detergents into biochemistry are retired (e.g., the 
Reynolds) or moved on to other things (e.g., Helenius).  Lastly, even 
when academics measure the properties of a detergent, the work is not 
generally published, aside from being buried deeply in a thesis or is 
so specific to certain conditions that the information may not be very 
useful.


One alternative is to enlist Anatrace and its parent USB, for example, 
to set up a Wiki for depositing detergent information and notes.  
Thus, anyone who has primary data on detergents could place it there 
with relevant experimental details.  In the spirit of full disclosure, 
I still consult with Anatrace and have provided them with some of my 
group's primary data on detergents. 


Best regards,

Michael


//

/R. Michael Garavito, Ph.D./

/Professor of Biochemistry & Molecular Biology/

/513 Biochemistry Bldg.   /

/Michigan State University  /

/East Lansing, MI 48824-1319/

/Office://  //(517) 355-9724 Lab:  (517) 353-9125/

/FAX:  (517) 353-9334Email:  [EMAIL PROTECTED] 
<mailto:[EMAIL PROTECTED]>/


//



On Oct 22, 2007, at 5:48 PM, Jacob Keller wrote:


Dear CCP4BB,

Although this is not exactly CCP4-related, I thought somebody here 
might know whether there is somewhere a definitive list or tabulation 
of detergent properties which are not simply copied out of catalogs, 
but have been traceably experimentally determined. In particular, it 
would be great to have phase diagrams for common detergents with 
detergent concentration versus pH, salt concentration, temperature, 
etc. Would this not be incredibly helpful for the scientific 
community? And yet, this is not so easy to find


Jacob


***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.467.4049
cel: 773.608.9185
email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
***





Re: [ccp4bb] questions about hydrophobic core

2007-06-19 Thread Nadir T. Mrabet

Hi,

There exists a formal analytical way in doing this using the "survol" 
command in BRUGEL package.
In short, this command define the accessible surface (external and 
cavities) to the probe you choose and creates

separate masks (ensembles) of all atoms/residues that define these surfaces.
The way to go, then, would be to create a collection mask of all 
accessible atoms/residues and subtract this
from your protein mask to be left with the mask that contains the core 
residues.
You could also use a cutoff of 5-10% ASA max (there are different ways 
of calculating these !).
On the other hand, taking away even residues that display very little 
ASA (< 5%) would certainly leave you with

genuine core residues.
Hope this helps.

Greetings,

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   UHP - Nancy 1, School of Medicine
   Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   Cell.: +33 (0)6.11.35.69.09


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Sebastiano Pasqualato wrote:


Hi all,
a few days ago I sent a post in which I was asking if anybody knew a 
program to automatically define the hydrophobic core of a protein, 
given the pdb.
Unfortunately I got no answers, and indeed a more thorough googling 
around revealed that such a program might not exist.

So it seems I have to define my hydrophobic core residues by hand...
So now my question would be: how to define the hydrophobic core residues?
I would tend to say that those that bury more than ## % (say 70%, 80% 
??) of their otherwise solvent accessible surface area could be 
defined as such, but how can I get such a /per residue/ percentage? 
(NB: this is not the asa buried upon interaction, so I don't know how 
to get the asa of the "free" amino acid)
Alternatively, are there other simple and defined rules to state which 
are the hydrophobic core residues?

Any help appreciated,
thanks in advance,
ciao
s



--
Sebastiano Pasqualato, PhD
IFOM-IEO Campus
Dipartimento di Oncologia Sperimentale
Istituto Europeo di Oncologia
via Adamello, 16
20139 Milano
Italy

tel +39 02 9437 5094
fax +39 02 574 303 310





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Re: [ccp4bb] Nature policy update regarding source code

2007-03-23 Thread Nadir T. Mrabet

Hi,

I believe such requirements concern only "Nature Methods" rather than 
"Nature" by and large.

Regards,

Nadir Mrabet

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   UHP - Nancy 1, School of Medicine
   Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   



[EMAIL PROTECTED] wrote:

I thought that some of you might be interested that the journal Nature
has clarified the publication requirements regarding source code
accessibility.  It is likely that some of you deserve congrats
for this.  Cheers!

http://www.nature.com/nmeth/journal/v4/n3/full/nmeth0307-189.html

Although there are still some small problems, I think that this is a
big step forward, and certainly an interesting read, if you are
interested in FOSS and science.

Regards,
Michael L. Love Ph.D
Department of Biophysics and Biophysical Chemistry
School of Medicine
Johns Hopkins University
725 N. Wolfe Street
Room 608B WBSB
Baltimore MD 21205-2185

Interoffice Mail: 608B WBSB, SoM

office: 410-614-2267
lab:410-614-3179
fax:410-502-6910
cell:   443-824-3451
http://www.gnu-darwin.org/



  


Re: [ccp4bb] PCB buffer and MMT buffer

2007-01-31 Thread Nadir T. Mrabet

Hi all,

I only wish to draw your attention to the fact that the 
Henderson-Hasselbach equation (as used in the paper you cite) is only 
valid if you use zwitterionic buffers. Otherwise, with other buffers, 
especially phosphate and multi-acids (e.g. succinate, citrate and so 
on), any calculation using the Henderson-Hasselbach equation must refer 
to activities and not concentrations.
Given this, expect a phosphate buffer pH to increase by 0.3-0.4 units 
upon a 10-fold dilution (That is from 1.0M to 0.1M in the referenced paper).


Regards,

Pr. Nadir T. Mrabet
   Cellular & Molecular Biochemistry
   INSERM U-724
   UHP - Nancy 1, School of Medicine
   Avenue de la Foret de Haye, BP 184
   54505 Vandoeuvre-les-Nancy Cedex
   France
   Phone: +33 (0)3.83.68.32.73
   Fax:   +33 (0)3.83.68.32.79
   E-mail: [EMAIL PROTECTED]
   Cell.: +33 (0)6.11.35.69.09


LEGAL NOTICE
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Jose M de Pereda wrote:

Hi Jenny (and others),

The buffer systems used in the PACT screen are described in the 
following paper by Janet Newman.


/Acta Cryst./ (2004). D*60*, 610-612[ 
doi:10.1107/S0907444903029640 
<http://dx.doi.org/10.1107/S0907444903029640> ]

Novel buffer systems for macromolecular crystallization
J. Newman 
<http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Newman,%20J.> 



I am not aware of any commercial source of these buffer systems. But 
the paper describes the recipes to make them, which is quite easy.


HTH

 Jose



On 30/01/2007 5:19 Jenny wrote:

Hi,

Sorry to bother you all.I'm going to try to reproduce some results
from the initial screening from PACT kit.The conditions are something
like 0.1M PCB buffer, 25% w/v PEG 1500 and 0.1 MMT buffer, 25%w/v PEG
1500. I was just wondering is there any easy way to get these
buffers?Do you happen to know where to order?Or I have to order the
component formula separately and then make buffers by myself?Thanks.

Jenny