[ccp4bb] on Rwork and Rfree

2012-02-07 Thread Dialing Pretty
Dear All,
 
After we refine the structure of the protein to satisfactory with 
satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree 
always increases slightly after the water picking refinment. 
 
Do you have nay idea to solve this problem or any comment?
 
Cheers,
 
Dialing

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Mark J van Raaij
Rationalising it completely may only be possible once you know the nature of 
the crystal contacts, i.e. when you have solved the structure. Until then it is 
mainly a matter of experimenting.
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Theresa H. Hsu
 Sent: Monday, February 06, 2012 11:00 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Freezing crystal
 
 Hi all
 
 Thanks for all the suggestions which I will try soon.
 
 How do the crystallization condition (PEG vs. salts like ammonium
 sulfate) affect the croyprotectant condition? Do factors like presence
 of low concentration of high molecular weight PEG ( 2000) mean PEG is
 better? Do buffers and salts in protein also important?
 
 Trying to rationalize it :)
 
 Theresa


Re: [ccp4bb] Fwd: tcl tk RHEL 6.2

2012-02-07 Thread Kay Diederichs

Hi,

I think your 64bit system lacks the 32bit loader. Try as root:

yum install /lib/ld-linux.so.2

and be prepared to install a couple of 32bit libraries, as needed.

HTH,

Kay




 Original Message 
Subject: tcl tk RHEL 6.2
Date: Mon, 6 Feb 2012 10:17:22 -0600
From: Kenneth A. Satyshur satys...@wisc.edu

I just installed ccp4 6.2.0 on a new RHEL6.2 system and I get this error.
Anyone know how to fix this? I installed tcl and tk into the system but
ccp4 seems to
want to use its own version.

[xray@paprika ~/Desktop]$
/home/xray/ccp4/ccp4-6.2.0/ccp4-6.2.0/bin/ccp4i:
/home/xray/ccp4/ccp4-6.2.0/tcltkplusplus/bin/wish: /lib/ld-linux.so.2:
bad ELF interpreter: No such file or directory
/home/xray/ccp4/ccp4-6.2.0/ccp4-6.2.0/bin/ccp4i: line 4:
/home/xray/ccp4/ccp4-6.2.0/tcltkplusplus/bin/wish: Success

thanks
kas


--
Kenneth A. Satyshur, M.S.,Ph.D.
Associate Scientist
University of Wisconsin
Madison, Wisconsin 53706
608-215-5207





--
Kay Diederichshttp://strucbio.biologie.uni-konstanz.de
email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz

This e-mail is digitally signed. If your e-mail client does not have the
necessary capabilities, just ignore the attached signature smime.p7s.



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Description: S/MIME Cryptographic Signature


[ccp4bb] mutagenesis based on ligand-bound crystal structures

2012-02-07 Thread Chris Ulens

Dear colleagues,
I am looking for a few example studies in which a complete mutagenesis  
of all residue-ligand interactions has been conducted to evaluate the  
energetic contributions of each individual interaction.

Thank you.
Best regards.
-Chris

---
Chris Ulens, Ph.D.
Lab of Structural Neurobiology
Department of Cellular and Molecular Medicine
Campus Gasthuisberg, ON1
Herestraat 49, PB 601
B-3000 Leuven
Belgium
e   chris.ul...@med.kuleuven.be
t+32 16 330689
f+32 16 345699
w   http://www.xtal.be


Re: [ccp4bb] mutagenesis based on ligand-bound crystal structures

2012-02-07 Thread Savvas Savvides
Dear Chris
A whole series of studies from the early to late 90's did his for the growth 
hormone-GHR interaction. 
Papers by Pearce, Cunningham, Clackson and Wells come to mind. 
Also a recent study on the IL13-IL13R interaction by Lupardus et al has covered 
most of the interaction epitope. 

Best regards
Savvas

On 07 Feb 2012, at 10:16, Chris Ulens chris.ul...@med.kuleuven.be wrote:

 Dear colleagues,
 I am looking for a few example studies in which a complete mutagenesis of all 
 residue-ligand interactions has been conducted to evaluate the energetic 
 contributions of each individual interaction.
 Thank you.
 Best regards.
 -Chris
 
 ---
 Chris Ulens, Ph.D.
 Lab of Structural Neurobiology
 Department of Cellular and Molecular Medicine
 Campus Gasthuisberg, ON1
 Herestraat 49, PB 601
 B-3000 Leuven
 Belgium
 e   chris.ul...@med.kuleuven.be
 t+32 16 330689
 f+32 16 345699
 w   http://www.xtal.be


Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Eleanor Dodson

On 02/07/2012 08:31 AM, Dialing Pretty wrote:

Dear All,

After we refine the structure of the protein to satisfactory with
satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree 
always increases slightly after the water picking refinment.

Do you have nay idea to solve this problem or any comment?

Cheers,

Dialing



Well - it shouldnt!

 Check if there is some problem..
Eleanor


Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Robbie Joosten
Hi Dialing,

 

Most water picking tools are rather overenthusiastic and end up placing some
waters at places where they should not be. This causes some overfitting and
an increase of R-free. I'm hideously old-fashioned and recommend
conservatively building waters by hand. 

There are some good validation tools that help get rid of excess water:
centrifuge in PDB_REDO does the basic work; check/delete waters in Coot
highlights other suspicious waters; WHAT_CHECK checks hydrogen bonding
thoroughly, finds nonsense clusters of water and also finds possible ions.
It must be noted that all these tools break down at very high resolution
where you may get alternate waters. Fortunately, this problem doesn't occur
very often.

 

Cheers,

Robbie

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Dialing Pretty
Sent: Tuesday, February 07, 2012 09:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] on Rwork and Rfree

 

Dear All,

 

After we refine the structure of the protein to satisfactory with
satisfactory Rwork and Rfree, we pick water by phenix refine, and I find
Rfree always increases slightly after the water picking refinment. 

 

Do you have nay idea to solve this problem or any comment?

 

Cheers,

 

Dialing

 

 

 



Re: [ccp4bb] shape complementarity between protein and DNA surface

2012-02-07 Thread Tim Gruene
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Deepak,

the only atom type present in DNA and not in Proteins would be the
Phosphorus. You can probably add this to sc_radii.lib yourself with the line
***P*  1.80

the radius stems from http://en.wikipedia.org/wiki/Phosphorus and the
link to http://en.wikipedia.org/wiki/Van_der_Waals_radius provides you
with the reference.

The results will hardly differ if you modify this value within 100%, I
guess.

Cheers,
Tim


On 02/07/2012 04:19 AM, Deepak Thankappan Nair wrote:
 Dear All
 For calculation of the shape complementarity index using the Sc
 program between a protein and DNA surface, does anybody have a
 modified radii.lib file that includes information about DNA atoms?
 Also, is there any reference that has information regarding the atomic
 radii of DNA atoms?
 thanks
 Deepak
 

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

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.10 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPMPu1UxlJ7aRr7hoRAhAYAKDHJzkHfEPwcHO99FAV57zOrsYasACgvO/V
CtUM1Z8x0NSl0cXK4A4tH0k=
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[ccp4bb] Fwd: TR: Permanent Scientist at beamline SWING

2012-02-07 Thread Louis RENAULT

Hi,
I am forwarding an announcement for a permanent scientist position in 
structural biology using SAXS

at the synchrotron SOLEIL located in Paris suburbs, France.
Please address all questions concerning the position to the responsible 
: Dr. Javier PEREZ pe...@synchrotron-soleil.fr

Best, Louis R


 Original Message 
Subject:TR: Permanent Scientist at beamline SWING
Date:   Wed, 1 Feb 2012 14:40:04 +
From:   PEREZ Javier pe...@synchrotron-soleil.fr
To: PEREZ Javier pe...@synchrotron-soleil.fr


Dear User,

Please find an announcement for permanent scientist position in 
structural biology
at beamline SWING dedicated to Small Angle X-ray Scattering (SAXS)on the 
synchrotron SOLEIL.

You'll find on the following link the conditions of the offer.

http://www.synchrotron-soleil.fr/portal/page/portal/Soleil/OffresEmplois/Scientifique-ligne-SWING-CDI
(click on the bristish flag if it is not displayed in english)


Best regards,

Javier Perez

*Javier Pérez*

Group leader

Beamline SWING

*Synchrotron SOLEIL*

L'Orme des Merisiers

Saint-Aubin - BP 48

91192 Gif sur Yvette Cedex

France

Tel : +33 (0)1 69 35 96 19 (office)

+33 (0)1 69 35 97 53 (beamline)

*javier.pe...@synchrotron-soleil.fr 
mailto:javier.pe...@synchrotron-soleil.fr*




[ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Deepak Oswal
Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a
catalytically critical aspartic acid has increased to 6.44. It is hydrogen
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
proton during the catalysis. Can anybody help me a) interpret the
significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
in context with the catalysis? Is this advantageous or detrimental? b) How
is pKa related to an amino acids’ ability to force a water molecule to
donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
similar increase in pKa values observed for aspartic acids before? I would
be grateful if anybody could explain or comment on the above queries.

Deepak Oswal


Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

2012-02-07 Thread Fred

Hi CCP4 list,
Thank you very much for additional messages and references.
Here goes the image of the PCR product before digested and after 
digested and cleaned.

 http://ompldr.org/vY29jbA
The results of the transformation of 3 microL (90 ng) of 
non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 
digested sample gave two colonies only; and transformation of 3 
microL(90 ng) of cleaned product gave 14 colonies.
So, if the amplification is not abundant, chances are that home made 
competent cell will not be transformed with the digested product. Don't 
want start another discussion but, is there any reason for differences 
in the transformation efficiency between the parental and the mutated 
(cleaned) plasmids?

All the Best,
Fred

Em 03-02-2012 16:14, Fred escreveu:

Hi CCP4 list,
Thanks everyone who have answered my post concerning to mutagenesis.
From quick reading most of the answers, the following seems to be a 
consensus:

1) Do not concentrate your PCR product;
2) Too much DNA and/or impurities like salts or whatever can inhibits 
transformation;
3) Purify your PCR product before transformation if possible or use 3 
of 4 microL of it. This is more or less the amount of DNA showed in 
the uploaded image.

Kind regards,
Fred

P.S.: I'll let you know the results.


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Clement Angkawidjaja

Hi Deepak,

Assuming that you have done the necessary things to measure the pKr of 
that particular Asp, I would say that the increase is advantageous for 
your enzyme. Enzyme catalysis often involves very subtle changes on the 
ionization state of the active site. But you need to be very careful in 
proposing a catalysis mechanism.


b) How is pKa related to an amino acids’ ability to force a water 
molecule to donate a proton?
Are you sure that the water donates a proton? Was your resolution high 
enough to observe it? How did you measure that pKr, by the way?


c) At pH 7.4, the aspartic acid would be de-protonated irrespective of 
whether the pKa is 3.8 or 6.44; isn’t that true?
I would say that the dominant fraction of Asp is deprotonated. But as 
you can see in the papers below, the pKr of Asp can vary from 0.5 to 9.2 
in folded proteins.


d) Have similar increase in pKa values observed for aspartic acids before?
there are some papers by Nick Pace's group:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708032/?tool=pubmed
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679426/?tool=pubmed
http://www.sciencedirect.com/science/article/pii/S002228360600934X

Cheers,
Clement

--


On 2/7/12 8:48 PM, Deepak Oswal wrote:


Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is 
hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed 
to donate a proton during the catalysis. Can anybody help me a) 
interpret the significance of this increase in pKa of the aspartic 
acid from 3.8 to 6.44 in context with the catalysis? Is this 
advantageous or detrimental? b) How is pKa related to an amino acids’ 
ability to force a water molecule to donate a proton? c) At pH 7.4, 
the aspartic acid would be de-protonated irrespective of whether the 
pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa 
values observed for aspartic acids before? I would be grateful if 
anybody could explain or comment on the above queries.


Deepak Oswal






Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jacob Keller
One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try

JPK

On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij mjvanra...@cnb.csic.es wrote:
 Rationalising it completely may only be possible once you know the nature of 
 the crystal contacts, i.e. when you have solved the structure. Until then it 
 is mainly a matter of experimenting.

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Theresa H. Hsu
 Sent: Monday, February 06, 2012 11:00 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Freezing crystal

 Hi all

 Thanks for all the suggestions which I will try soon.

 How do the crystallization condition (PEG vs. salts like ammonium
 sulfate) affect the croyprotectant condition? Do factors like presence
 of low concentration of high molecular weight PEG ( 2000) mean PEG is
 better? Do buffers and salts in protein also important?

 Trying to rationalize it :)

 Theresa



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

2012-02-07 Thread Xiaodi Yu

Hi Fred:

For the mutated plasmids, it generates the nicked dna, so the transform 
efficiency will be lower compared to the parental plasmids. And that is the 
reason why people usually use super competent cell to transform these nicked 
plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You 
just need ONE. And usually for these mutagenesis PCR, the success rate is 
pretty high. I suggest you to sequence these colonies. And you can get it.

Yu Xiaodi

 Date: Tue, 7 Feb 2012 10:17:43 -0200
 From: ccp4bb.l...@gmail.com
 Subject: Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis 
 [OFF-TOPIC]
 To: CCP4BB@JISCMAIL.AC.UK
 
 Hi CCP4 list,
 Thank you very much for additional messages and references.
 Here goes the image of the PCR product before digested and after 
 digested and cleaned.
   http://ompldr.org/vY29jbA
 The results of the transformation of 3 microL (90 ng) of 
 non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 
 digested sample gave two colonies only; and transformation of 3 
 microL(90 ng) of cleaned product gave 14 colonies.
 So, if the amplification is not abundant, chances are that home made 
 competent cell will not be transformed with the digested product. Don't 
 want start another discussion but, is there any reason for differences 
 in the transformation efficiency between the parental and the mutated 
 (cleaned) plasmids?
 All the Best,
 Fred
 
 Em 03-02-2012 16:14, Fred escreveu:
  Hi CCP4 list,
  Thanks everyone who have answered my post concerning to mutagenesis.
  From quick reading most of the answers, the following seems to be a 
  consensus:
  1) Do not concentrate your PCR product;
  2) Too much DNA and/or impurities like salts or whatever can inhibits 
  transformation;
  3) Purify your PCR product before transformation if possible or use 3 
  of 4 microL of it. This is more or less the amount of DNA showed in 
  the uploaded image.
  Kind regards,
  Fred
 
  P.S.: I'll let you know the results.
  

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Xiaodi Yu

Hi Deepak:

I think it is common for the residues which participate catalysis to have a Pka 
deviated from the reality pKa value especially for acid/base catalysis (acid 
base titration assay can help you to figure out the way of catalysis). Usually 
the pKa values of these kind of critical residues are affected by their local 
environment and this character is related to the enzyme's working mechanism.

I am sorry that I am not professional in enzyme, I cannot answer your questions 
for each questions.

Yu Xiaodi

Date: Tue, 7 Feb 2012 19:48:26 +0800
From: deepos...@gmail.com
Subject: [ccp4bb] On pKa of Aspartic acid
To: CCP4BB@JISCMAIL.AC.UK


Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is hydrogen 
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton 
during the catalysis. Can anybody help me a) interpret the significance of this 
increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the 
catalysis? Is this advantageous or detrimental? b) How is pKa related to an 
amino acids’ ability to force a water molecule to donate a proton? c) At pH 
7.4, the aspartic acid would be de-protonated irrespective of whether the pKa 
is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values 
observed for aspartic acids before? I would be grateful if anybody could 
explain or comment on the above queries.

Deepak Oswal  

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Roger Rowlett

  
  
Residue pKa values in proteins are strongly
  affected by the local environment and can deviate far from the
  "norm". Of course, the higher the pKa of the residue, the stronger
  general base it will be. There is a significant
  thermodynamic/kinetic advantage in matching the pKa of a general
  base to the pKa of the proton that must be removed for catalysis.
Aspartate frequently plays a role as a general base for assisting
water in nucleophilic attack.

I think it is unusual for water to act as a general acid (the pKa of
water is 14-15, depending on how you calculate it), but if that is
the case and there is compelling evidence that water is the proton
donor in your reaction, it would be necessary for aspartic acid to
be in its protonated form in order to assist a water-mediated
protonation event. Raising the pKa of aspartic acid would allow a
larger fraction of it to be in its protonated state at
physiologically relevant pH values, although it would reduce the
intrinsic effectiveness of Asp as a general acid. There should be a
significant thermodynamic and kinetic advantage in having Asp
participate directly in a general acid catalyzed reaction, rather
than through a water molecule.

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 2/7/2012 10:00 AM, Xiaodi Yu wrote:

  
  
Hi Deepak:

I think it is common for the residues which participate
catalysis to have a Pka deviated from the reality pKa value
especially for acid/base catalysis (acid base titration assay
can help you to figure out the way of catalysis). Usually the
pKa values of these kind of critical residues are affected by
their local environment and this character is related to the
enzyme's working mechanism.

I am sorry that I am not professional in enzyme, I cannot answer
your questions for each questions.

Yu Xiaodi


  Date: Tue, 7 Feb 2012 19:48:26 +0800
  From: deepos...@gmail.com
  Subject: [ccp4bb] On pKa of Aspartic acid
  To: CCP4BB@JISCMAIL.AC.UK
  
  
Dear colleagues,
We have solved
the crystal structure of a human enzyme. The pKa of a
catalytically critical aspartic acid has increased to
6.44. It is hydrogen bonded (2.8 Angstroms) to a water
molecule that is supposed to donate a proton during the
catalysis. Can anybody help me a) interpret the
significance of this increase in pKa of the aspartic
acid from 3.8 to 6.44 in context with the catalysis? Is
this advantageous or detrimental? b) How is pKa related
to an amino acids’ ability to force a water molecule to
donate a proton? c) At pH 7.4, the aspartic acid would
be de-protonated irrespective of whether the pKa is 3.8
or 6.44; isn’t that true? d) Have similar increase in
pKa values observed for aspartic acids before? I would
be grateful if anybody could explain or comment on the
above queries.
Deepak Oswal
  
  

  



Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
Something to add into this discussion is also go fro the tiny crystals versus 
the big ones.
BIGGER is not always BETTER - in particular if you try to freeze directly out 
of your conditions without an additional cryo-protectant. And with small or 
tiny I mean 10 micron, whatever you are capable of mounting. It is also 
important to keep the amount of liquid volume around the crystal low, so rather 
use a loop in which you scoop the crystal up instead of having a large loop 
with lots of liquid.

Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a 
quicker freeze of your material.

If you have the option to anneal your crystal after testing it in the beam try 
it out and assess the success or damage, this will be very different depending 
on what cryo-additives you have around.

Good luck,

Jürgen

On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote:

One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try

JPK

On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij 
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote:
Rationalising it completely may only be possible once you know the nature of 
the crystal contacts, i.e. when you have solved the structure. Until then it is 
mainly a matter of experimenting.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Theresa H. Hsu
Sent: Monday, February 06, 2012 11:00 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Freezing crystal

Hi all

Thanks for all the suggestions which I will try soon.

How do the crystallization condition (PEG vs. salts like ammonium
sulfate) affect the croyprotectant condition? Do factors like presence
of low concentration of high molecular weight PEG ( 2000) mean PEG is
better? Do buffers and salts in protein also important?

Trying to rationalize it :)

Theresa



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
***

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/






Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Pavel Afonine
Dialing,


After we refine the structure of the protein to satisfactory with
 satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree
 always increases slightly after the water picking refinment.

 Do you have nay idea to solve this problem or any comment?


Just a reminder: there is phenixbb mailing list for Phenix related
questions.

Replying to your post: phenix.refine uses very sophisticated criteria for
water update (which doen't mean there is no room for further improvement of
course). If you believe it placed or removed a wrong water please submit a
bug report to me that should include:
1) all input files (reflection data, PDB file with the model, CIF ligand
and parameter files if any);
2) the command you used to run the program;
3) explain which waters you believe are wrong.

Pavel.





Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
should be 50/50.
In this case, we may need to consider water in two formats:

H2O vs. H3O+

When we say water as acid, it usually stands for H3O+ in chemistry. In
chemical equation, H+ represents H3O+.

In enzyme catalysis, water as a general acid sounds reasonable under
pH 7.0. In some famous paper, water has been concluded as the general
base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
to be reconsidered.
.
Recently, I have read papers for pKa perturbation. I am also
interested in the general base of Asp and Glu in enzyme catalysis.


I will be very happy to read your paper in the future.

Regards,

Kevin Jin

On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 Dear colleagues,

 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.

 Deepak Oswal


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Fischmann, Thierry
Check this review, for instance:

Pace, C. et al. Protein Ionizable Groups:  pK values and Their Contribution to 
Protein Stability and Solubility.  J. Biol Chem.  284, 13285-13289 (May 15, 
2009)

Thierry



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu
Sent: Tuesday, February 07, 2012 10:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] On pKa of Aspartic acid

Hi Deepak:

I think it is common for the residues which participate catalysis to have a Pka 
deviated from the reality pKa value especially for acid/base catalysis (acid 
base titration assay can help you to figure out the way of catalysis). Usually 
the pKa values of these kind of critical residues are affected by their local 
environment and this character is related to the enzyme's working mechanism.

I am sorry that I am not professional in enzyme, I cannot answer your questions 
for each questions.

Yu Xiaodi


Date: Tue, 7 Feb 2012 19:48:26 +0800
From: deepos...@gmail.com
Subject: [ccp4bb] On pKa of Aspartic acid
To: CCP4BB@JISCMAIL.AC.UK


Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is hydrogen 
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton 
during the catalysis. Can anybody help me a) interpret the significance of this 
increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the 
catalysis? Is this advantageous or detrimental? b) How is pKa related to an 
amino acids' ability to force a water molecule to donate a proton? c) At pH 
7.4, the aspartic acid would be de-protonated irrespective of whether the pKa 
is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values 
observed for aspartic acids before? I would be grateful if anybody could 
explain or comment on the above queries.

Deepak Oswal
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] On pKa of Aspartic acid

2012-02-07 Thread Fischmann, Thierry
Oops, you meant catalytic residue. Check the following:

Harris TK  Turner GJ Structural basis of pertubed pKa values of catalytic 
groups in enzyme active sites IUBMB life, 53 85-98 (Feb 2002)

Thierry


From: Fischmann, Thierry
Sent: Tuesday, February 07, 2012 10:59 AM
To: 'Xiaodi Yu'; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] On pKa of Aspartic acid

Check this review, for instance:

Pace, C. et al. Protein Ionizable Groups:  pK values and Their Contribution to 
Protein Stability and Solubility.  J. Biol Chem.  284, 13285-13289 (May 15, 
2009)

Thierry



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu
Sent: Tuesday, February 07, 2012 10:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] On pKa of Aspartic acid

Hi Deepak:

I think it is common for the residues which participate catalysis to have a Pka 
deviated from the reality pKa value especially for acid/base catalysis (acid 
base titration assay can help you to figure out the way of catalysis). Usually 
the pKa values of these kind of critical residues are affected by their local 
environment and this character is related to the enzyme's working mechanism.

I am sorry that I am not professional in enzyme, I cannot answer your questions 
for each questions.

Yu Xiaodi


Date: Tue, 7 Feb 2012 19:48:26 +0800
From: deepos...@gmail.com
Subject: [ccp4bb] On pKa of Aspartic acid
To: CCP4BB@JISCMAIL.AC.UK


Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is hydrogen 
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton 
during the catalysis. Can anybody help me a) interpret the significance of this 
increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the 
catalysis? Is this advantageous or detrimental? b) How is pKa related to an 
amino acids' ability to force a water molecule to donate a proton? c) At pH 
7.4, the aspartic acid would be de-protonated irrespective of whether the pKa 
is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values 
observed for aspartic acids before? I would be grateful if anybody could 
explain or comment on the above queries.

Deepak Oswal
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] Freezing crystal

2012-02-07 Thread Enrico Stura

BIGGER is not always BETTER?

Theoretically it should be better because you have more scattering matter.  
If it is

not something has gone wrong in prior steps:
Purification: You were less selective and picked up more heterogeneous
protein.
Crystallization: The bigger crystals grew under conditions that were less
controlled because of changes in the state of protein supersaturation,  
precipitant or
temperature. These inhomogeneities contributed to give you bigger, but not  
better

crystals.
Cryo-soak: Bigger crystals are more prone to be shocked when transfered to  
a
less than optimal cryo-solution. This is a critical step, and crystals do  
not like too
much the cryo-chemicals. To test this I tried lower concentrations of  
various

cryo-compounds instead of a huge quantity of a single component and in most
cases the mixture was better tollerated than the single components.
Flash-freezing: Bigger crystals will cool more unevenly than small ones. A  
change from
liquid nitrogen to liquid ethane could achieve faster cooling because of  
the

greater heat capacity of the latter liquid.

Large crystals are more difficult to handle than small ones but after  
experimenting with
small ones we can build up a good experimental protocol so that the big  
crystals will give exceptionally
good results. I compared small crystals on high intensity beamlines at the  
ESRF against
large crystals on BM30 and the big crystals were statistically better.  
Unfortunately, it takes
a lot of time and a certain amount of expertize to optimize the  
conditions. So ...  although I

strongly disagree with Jürgen, I will also advise to start with small ones.

Enrico.

On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen jubo...@jhsph.edu  
wrote:


Something to add into this discussion is also go fro the tiny crystals  
versus the big ones.
BIGGER is not always BETTER - in particular if you try to freeze  
directly out of your conditions without an additional cryo-protectant.  
And with small or tiny I mean 10 micron, whatever you are capable of  
mounting. It is also important to keep the amount of liquid volume  
around the crystal low, so rather use a loop in which you scoop the  
crystal up instead of having a large loop with lots of liquid.


Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2  
leads to a quicker freeze of your material.


If you have the option to anneal your crystal after testing it in the  
beam try it out and assess the success or damage, this will be very  
different depending on what cryo-additives you have around.


Good luck,

Jürgen

On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote:

One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try

JPK

On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij  
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote:
Rationalising it completely may only be possible once you know the  
nature of the crystal contacts, i.e. when you have solved the structure.  
Until then it is mainly a matter of experimenting.


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Theresa H. Hsu
Sent: Monday, February 06, 2012 11:00 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Freezing crystal

Hi all

Thanks for all the suggestions which I will try soon.

How do the crystallization condition (PEG vs. salts like ammonium
sulfate) affect the croyprotectant condition? Do factors like presence
of low concentration of high molecular weight PEG ( 2000) mean PEG is
better? Do buffers and salts in protein also important?

Trying to rationalize it :)

Theresa



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
***

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/







--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Christian Roth
Hi,

you may also look into the papers of John A. Gerlt, who did a lot on 
protonabtraction reactions and the theory behind this. Esspecially the pKa 
disturbance and the match to the pkA of the substrate of the reaction. 

Best Wishes 

Christian

Am Dienstag 07 Februar 2012 12:48:26 schrieb Deepak Oswal:
 Dear colleagues,
 
 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.
 
 Deepak Oswal
 


Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
Hi Enrico,

I was just looking at non-optimal cryo-conditions and the original posters 
starting point.
Of course if you have a good cryo bigger is better for the reasons you write 
but if you have no clue how your crystals will perform then I'd rather go for 
small to be cautious and also have those around and not only the big ones which 
everybody mounts because they looks so nice. To be disappointed by big crystals 
is often not a surprise to me and if you have not tried small crystals from the 
same batch well then you missed 50% of your chances to solve s structure with 
the first light the crystals saw.

Jürgen



On Feb 7, 2012, at 11:51 AM, Enrico Stura wrote:

BIGGER is not always BETTER?

Theoretically it should be better because you have more scattering matter.
If it is
not something has gone wrong in prior steps:
Purification: You were less selective and picked up more heterogeneous
protein.
Crystallization: The bigger crystals grew under conditions that were less
controlled because of changes in the state of protein supersaturation,
precipitant or
temperature. These inhomogeneities contributed to give you bigger, but not
better
crystals.
Cryo-soak: Bigger crystals are more prone to be shocked when transfered to
a
less than optimal cryo-solution. This is a critical step, and crystals do
not like too
much the cryo-chemicals. To test this I tried lower concentrations of
various
cryo-compounds instead of a huge quantity of a single component and in most
cases the mixture was better tollerated than the single components.
Flash-freezing: Bigger crystals will cool more unevenly than small ones. A
change from
liquid nitrogen to liquid ethane could achieve faster cooling because of
the
greater heat capacity of the latter liquid.

Large crystals are more difficult to handle than small ones but after
experimenting with
small ones we can build up a good experimental protocol so that the big
crystals will give exceptionally
good results. I compared small crystals on high intensity beamlines at the
ESRF against
large crystals on BM30 and the big crystals were statistically better.
Unfortunately, it takes
a lot of time and a certain amount of expertize to optimize the
conditions. So ...  although I
strongly disagree with Jürgen, I will also advise to start with small ones.

Enrico.

On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen 
jubo...@jhsph.edumailto:jubo...@jhsph.edu
wrote:

Something to add into this discussion is also go fro the tiny crystals
versus the big ones.
BIGGER is not always BETTER - in particular if you try to freeze
directly out of your conditions without an additional cryo-protectant.
And with small or tiny I mean 10 micron, whatever you are capable of
mounting. It is also important to keep the amount of liquid volume
around the crystal low, so rather use a loop in which you scoop the
crystal up instead of having a large loop with lots of liquid.

Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2
leads to a quicker freeze of your material.

If you have the option to anneal your crystal after testing it in the
beam try it out and assess the success or damage, this will be very
different depending on what cryo-additives you have around.

Good luck,

Jürgen

On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote:

One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try

JPK

On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij
mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es
 wrote:
Rationalising it completely may only be possible once you know the
nature of the crystal contacts, i.e. when you have solved the structure.
Until then it is mainly a matter of experimenting.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Theresa H. Hsu
Sent: Monday, February 06, 2012 11:00 PM
To: 
CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Freezing crystal

Hi all

Thanks for all the suggestions which I will try soon.

How do the crystallization condition (PEG vs. salts like ammonium
sulfate) affect the croyprotectant condition? Do factors like presence
of low concentration of high molecular weight PEG ( 2000) mean PEG is
better? Do buffers and salts in protein also important?

Trying to rationalize it :)

Theresa



--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: 
j-kell...@northwestern.edumailto:j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
***

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Hong Zhang
Is the water molecule in question coordinated to any other group(s)? 


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kevin
Jin
Sent: Tuesday, February 07, 2012 10:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] On pKa of Aspartic acid

As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
should be 50/50.
In this case, we may need to consider water in two formats:

H2O vs. H3O+

When we say water as acid, it usually stands for H3O+ in chemistry. In
chemical equation, H+ represents H3O+.

In enzyme catalysis, water as a general acid sounds reasonable under
pH 7.0. In some famous paper, water has been concluded as the general
base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
to be reconsidered.
.
Recently, I have read papers for pKa perturbation. I am also
interested in the general base of Asp and Glu in enzyme catalysis.


I will be very happy to read your paper in the future.

Regards,

Kevin Jin

On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 Dear colleagues,

 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids' ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn't that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.

 Deepak Oswal


[ccp4bb] Merging hi and low res data

2012-02-07 Thread Nicholas Keep

We have a high and low res pass on a crystal that is going to around 1.2 A on 
the pilatus at Diamond.
We tried to scale them together using the xds CORRECT files from the xia2 -3dii runs (gives best results as far as we 
can see comparing xia2.html files)  through aimless via ccp4i interface.


The combined dataset looks worse (based on merging statistics at lower resolutions) than the individual particular at 
low res.(Compared to scala xia2 runs).

XDS does not record the pilatus as having overloads even on the high res pass 
using the correct detector parameters
http://xds.mpimf-heidelberg.mpg.de/html_doc/detectors.html

What I wanted to do was limit the resolution of the two runs ie to use low res between 100-2.5 beyond which completeness 
plummets and then the highres between 8 and 1.2.  Aimless does not seem to allow me to do that at least by the ccp4i 
interface- is this then bad practice?  I guess I could run a utility to remove the resolutions I don't want.


The one effect that makes me wonder if the lowres is better data, and worth combining, ie there is some overload in the 
high res, is that it goes from 30-8 mean I/SIGI whereas the high res has I/SIGI of 15 in most of the low res shells and 
then slowly falls off to 2.0 at 1.2 angstrom.


Am intending to carry on just using the hires pass.

Any comments?

I think Phil is in NZ and not looking at email hence putting this to the whole 
community
--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G57 Office)
  020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the crystallography 
entrance
and ring me or the department office from the internal phone by the door


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
Maybe you would also be interested in

http://www.jinkai.org/AAD_history.html

Regards,

Kevin

On Tue, Feb 7, 2012 at 8:52 AM, Christian Roth
christian.r...@bbz.uni-leipzig.de wrote:
 Hi,

 you may also look into the papers of John A. Gerlt, who did a lot on
 protonabtraction reactions and the theory behind this. Esspecially the pKa
 disturbance and the match to the pkA of the substrate of the reaction.

 Best Wishes

 Christian

 Am Dienstag 07 Februar 2012 12:48:26 schrieb Deepak Oswal:
 Dear colleagues,

 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.

 Deepak Oswal



Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Dirk Kostrewa

Dear Jürgen,

Am 07.02.12 16:58, schrieb Bosch, Juergen:
snip
Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 
leads to a quicker freeze of your material.

/snip

Are you sure? There was a publication by Warkentin et al. [1] about a 
cold gas layer above liquid nitrogen that reduces the expected cooling 
rate a lot!
My very personal experience is, that cryo-cooling in the N2-stream 
worked better for me than in LN2 in a variety of projects - but the 
reason could just be me ;-)


Best regards,

Dirk.

[1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert 
E Thorne: Hyperquenching for protein cryocrystallography, J. Appl. 
Crystallogr., 39, 805-811 (2006)


--

***
Dirk Kostrewa
Gene Center Munich
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***


Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
Hi Dirk,

I remember a neat paper don't recall who wrote it. I think it was in Acta D 
where the authors made a tiny probe the size of an elongated crystal glued to a 
[/Advertisement on] Hampton loop [/Advertisement off]. The probe was a 
temperature sensor and they recorded the cooling rate under different methods. 
The winner as far as I recall was freezing in liquid propane for the lack of 
the missing gas layer, but the second best method was LN2. Propane for whatever 
reason has gone extinct in certain areas of the world :-) . I'll try to find 
that reference but perhaps somebody else on this highly educated board knows 
which paper I'm referring to. I want to say it was published around 2004-2006.

Jürgen

On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote:

Dear Jürgen,

Am 07.02.12 16:58, schrieb Bosch, Juergen:
snip
Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2
leads to a quicker freeze of your material.
/snip

Are you sure? There was a publication by Warkentin et al. [1] about a
cold gas layer above liquid nitrogen that reduces the expected cooling
rate a lot!
My very personal experience is, that cryo-cooling in the N2-stream
worked better for me than in LN2 in a variety of projects - but the
reason could just be me ;-)

Best regards,

Dirk.

[1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert
E Thorne: Hyperquenching for protein cryocrystallography, J. Appl.
Crystallogr., 39, 805-811 (2006)

--

***
Dirk Kostrewa
Gene Center Munich
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.demailto:kostr...@genzentrum.lmu.de
WWW: www.genzentrum.lmu.de
***

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/






Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Francisco Hernandez-Guzman
Hi Deepak,

With regards observed pKa shifts, Prof. Ondrechen from Northeastern University 
has had a long interest in this field.

http://www.northeastern.edu/org/wp/

Under the computational tools that she has developed a program called THEMATICS 
that allows you to predict the pka of titratable amino acids and she has been 
able to predict shifts. Though the server seems to be down at this point, here 
is the reference: Y. Wei, J. Ko, L.F. Murga, and M.J. Ondrechen, BMC 
Bioinformatics 8:119, (2007)

From the commercial side, Dr. Spassov from Accelrys has also been working on 
tools that predict protein ionization. In his work, he has also been able to 
predict significant pka shifts for functionally relevant residues.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2578799/

Cheers,

Francisco


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Deepak 
Oswal
Sent: Tuesday, February 07, 2012 3:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] On pKa of Aspartic acid

Dear colleagues,
We have solved the crystal structure of a human enzyme. The pKa of a 
catalytically critical aspartic acid has increased to 6.44. It is hydrogen 
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton 
during the catalysis. Can anybody help me a) interpret the significance of this 
increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the 
catalysis? Is this advantageous or detrimental? b) How is pKa related to an 
amino acids' ability to force a water molecule to donate a proton? c) At pH 
7.4, the aspartic acid would be de-protonated irrespective of whether the pKa 
is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values 
observed for aspartic acids before? I would be grateful if anybody could 
explain or comment on the above queries.
Deepak Oswal


Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jim Pflugrath
Just a thought for those that mentioned propane and ethane, I would like to 
suggest that they try carbon tetrafluoride (CF4) instead.  It certainly should 
be much safer.  It melts at 90 K and boils at 145 K, so you know you are below 
145 K if you see it as a liquid.




Re: [ccp4bb] Freezing crystal

2012-02-07 Thread David Schuller

On 02/07/12 11:12, Dirk Kostrewa wrote:

Dear Jürgen,

Am 07.02.12 16:58, schrieb Bosch, Juergen:
snip
Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 
leads to a quicker freeze of your material.

/snip

Are you sure? There was a publication by Warkentin et al. [1] about a 
cold gas layer above liquid nitrogen that reduces the expected cooling 
rate a lot!
My very personal experience is, that cryo-cooling in the N2-stream 
worked better for me than in LN2 in a variety of projects - but the 
reason could just be me ;-)
Yes, Warkentin, et al found the cold gas layer affects cooling rate - 
but they were able to overcome it by blowing air across the surface. 
Your point is taken that complications such as this affect the cooling 
rate much more than a simple liquid vs. stream choice.



--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu


Re: [ccp4bb] Freezing crystal (Liquid Propane Crystal Prep)

2012-02-07 Thread Steven Herron


Jürgen Quote: Propane for whatever reason has gone extinct in certain 
areas of the world :-) .



I went to SSRL (Stanford) with a colleague who wanted to use liquid 
propane.  We had to go through a mound of paper work to get permission 
bring propane on site and set up the experiments.  I don't blame SSRL 
for their safety policy, but I can clearly understand why liquid propane 
is not commonly used.


If you don't think it is much of a danger, you might enjoy: 
http://www.stupidvideos.com/video/stunts/propane_tank/#2974
You might also enjoy:  
http://www.stupidvideos.com/video/stunts/C4_Propane_Explosion/#175408

 Note:  We did not bring any C-4 to SSRL:)

Steve



On 2/7/2012 10:50 AM, Bosch, Juergen wrote:

Hi Dirk,

I remember a neat paper don't recall who wrote it. I think it was in 
Acta D where the authors made a tiny probe the size of an elongated 
crystal glued to a [/Advertisement on] Hampton loop [/Advertisement 
off]. The probe was a temperature sensor and they recorded the cooling 
rate under different methods. The winner as far as I recall was 
freezing in liquid propane for the lack of the missing gas layer, but 
the second best method was LN2. Propane for whatever reason has gone 
extinct in certain areas of the world :-) . I'll try to find that 
reference but perhaps somebody else on this highly educated board 
knows which paper I'm referring to. I want to say it was published 
around 2004-2006.


Jürgen

On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote:


Dear Jürgen,

Am 07.02.12 16:58, schrieb Bosch, Juergen:
snip

Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2
leads to a quicker freeze of your material.

/snip

Are you sure? There was a publication by Warkentin et al. [1] about a
cold gas layer above liquid nitrogen that reduces the expected cooling
rate a lot!
My very personal experience is, that cryo-cooling in the N2-stream
worked better for me than in LN2 in a variety of projects - but the
reason could just be me ;-)

Best regards,

Dirk.

[1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert
E Thorne: Hyperquenching for protein cryocrystallography, J. Appl.
Crystallogr., 39, 805-811 (2006)

--

***
Dirk Kostrewa
Gene Center Munich
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail:kostr...@genzentrum.lmu.de mailto:kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/




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[ccp4bb] Staff scientist position at MacCHESS

2012-02-07 Thread Marian Szebenyi
This job was posted earlier, but there is an update to the how-to-apply 
instructions.


The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron
Source (MacCHESS) has an opening for a Staff Scientist (Research Associate) to
pursue the development of novel techniques in x-ray scattering as applied to
structural biology, and to support users at MacCHESS.  There will also be
opportunities to pursue projects in structural biology, using current
crystallographic and SAXS methods.  Research areas of particular interest
include structure solution from multiple crystals, use of Laue diffraction,
BioSAXS, microfluidics, and user interfaces for beamline operation.  A Ph.D. in
structural biology, biophysics, or a related field, and at least 3 years of
experience beyond the degree in a relevant field is required.  A solid
publication record is essential, and experience working at a synchrotron
facility is highly desirable.  Excellent communication skills are a must,
including fluency in the English language.  Appointments are nominally for three
years with the possibility for renewal, subject to mutual satisfaction and the
availability of funds.

Located on an Ivy League university campus in picturesque upstate New York, the
Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user base of
structural biologists, chemists, physicists, and engineers.  MacCHESS is an
NIH-supported National Resource providing support for structural biology at
CHESS.  MacCHESS is a heavily team-oriented environment.

Applications should be submitted at http://academicjobsonline.org/ (posting no. 
1422) and should include a cover letter, a CV, a list of publications, and a 
detailed summary of research experience and interests.  Applicants must arrange 
to have at least three letters of recommendation sent, as per instructions on 
the academicjobsonline website. The job is available immediately.


Cornell is an equal opportunity, affirmative action educator and employer.


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Zachary Wood
Hi Kevin,

Hate to point this out, but under pH 7.0, the protonation state of water is not 
50:50, and it is not a good acid.  The H30+ concentration of pure water is 
10^-7 Molar.  In pure water (assuming 55.5 M) only 1:555,000,000 water 
molecules is in the protonated, charged state (H3O+).  This is why when an 
enzyme uses water in its mechanism as a nucleophile, base, or acid, there is 
usually an acid/base catalyst or  metal that protonates or deprotonates the 
water to 'activate it'. 


Best regards,

Z


***
Zachary A. Wood, Ph.D.
Assistant Professor  
Department of Biochemistry  Molecular Biology
University of Georgia
Life Sciences Building, Rm A426B
120 Green Street
Athens, GA  30602-7229
Office: 706-583-0304
Lab:706-583-0303
FAX: 706-542-1738
***






On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote:

 As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
 should be 50/50.
 In this case, we may need to consider water in two formats:
 
 H2O vs. H3O+
 
 When we say water as acid, it usually stands for H3O+ in chemistry. In
 chemical equation, H+ represents H3O+.
 
 In enzyme catalysis, water as a general acid sounds reasonable under
 pH 7.0. In some famous paper, water has been concluded as the general
 base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
 hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
 to be reconsidered.
 .
 Recently, I have read papers for pKa perturbation. I am also
 interested in the general base of Asp and Glu in enzyme catalysis.
 
 
 I will be very happy to read your paper in the future.
 
 Regards,
 
 Kevin Jin
 
 On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 Dear colleagues,
 
 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.
 
 Deepak Oswal
 


Re: [ccp4bb] Merging hi and low res data

2012-02-07 Thread Kay Diederichs

Hi,

from your description I do not quite understand a few points:
- what is the evidence that there are overloads? The Pilatus pixels have 
20bits (meaning max contents of a pixel = 1048575) , and the OVERLOAD 
parameter is set to 1048500 (slightly below that). Do you actually see 
pixels with a contents of 1048575, but INTEGRATE and CORRECT do not 
report any overloads? That could indicate a bug in XDS, and I'd 
appreciate to receive a frame with overloads.
- could it be that the hi and the low resolution run do not scale well 
because they correspond to alternative indexing modes? - this can happen 
in spacegroups P3x, P4x, P6x and a couple of others. In that case you 
can use the XDS_ASCII.HKL of one run as a REFERENCE_DATA_SET for the 
other run, and run the CORRECT job - it will pick a consistent indexing.
- did you collect the hi res first, and the low res after that? If so, 
bad scaling is to be expected, because the crystal has a lot of 
radiation damage in the second run, resulting in elevated R values. It 
is much better to do the low res run first, producing very little 
radiation damage due to the much lower dose required.


best,
Kay


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
Oops, It should be: [H3O+]/[OH-]= 50/50

Kw = [H3O+][OH-],

pH = pKa +log ([OH-]/[H2O])

H3O+ concentration of pure water is 10^-7 mol/L

total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right?

Regards,

Kevin


On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu wrote:
 Hi Kevin,

 Hate to point this out, but under pH 7.0, the protonation state of water is 
 not 50:50, and it is not a good acid.  The H30+ concentration of pure water 
 is 10^-7 Molar.  In pure water (assuming 55.5 M) only 1:555,000,000 water 
 molecules is in the protonated, charged state (H3O+).  This is why when an 
 enzyme uses water in its mechanism as a nucleophile, base, or acid, there is 
 usually an acid/base catalyst or  metal that protonates or deprotonates the 
 water to 'activate it'.


 Best regards,

 Z


 ***
 Zachary A. Wood, Ph.D.
 Assistant Professor
 Department of Biochemistry  Molecular Biology
 University of Georgia
 Life Sciences Building, Rm A426B
 120 Green Street
 Athens, GA  30602-7229
 Office: 706-583-0304
 Lab:    706-583-0303
 FAX: 706-542-1738
 ***






 On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote:

 As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
 should be 50/50.
 In this case, we may need to consider water in two formats:

 H2O vs. H3O+

 When we say water as acid, it usually stands for H3O+ in chemistry. In
 chemical equation, H+ represents H3O+.

 In enzyme catalysis, water as a general acid sounds reasonable under
 pH 7.0. In some famous paper, water has been concluded as the general
 base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
 hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
 to be reconsidered.
 .
 Recently, I have read papers for pKa perturbation. I am also
 interested in the general base of Asp and Glu in enzyme catalysis.


 I will be very happy to read your paper in the future.

 Regards,

 Kevin Jin

 On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:
 Dear colleagues,

 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.

 Deepak Oswal




[ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Jacob Keller
Dear CCP4BB,

this is perhaps my most egregious off-topic post, but can anyone
explain why the following reference is not findable in PubMed? I can
get it from the ACS website, but not on PubMed or elsewhere. The
journal is on PubMed--is it perhaps because it's funded by ExxonMobil?
Very strange...

Jacob


Article
A Study of the Separation Principle in Size Exclusion Chromatography
AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240
KB]FiguresCiting Articles
Thomas Sun*
Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr.,
Baytown, Texas 77520-2101
Ronald R. Chance, William W. Graessley,† and David J. Lohse
Corporate Strategic Research, ExxonMobil Research and Engineering,
Annandale, New Jersey 08801
Macromolecules, 2004, 37 (11), pp 4304–4312
DOI: 10.1021/ma030586k
Publication Date (Web): April 29, 2004
Copyright © 2004 American Chemical Society

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Roger Rowlett

  
  
No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C.
  
  
So at pH 7.0, you have 10^-7 M each at
  equilibrium no matter how you slice it or whatever else is in
  solution. If equilibrium [H3O+] goes up [OH-] goes down
commensurately.

  The "pKa" of water as an acid is based on Kw and water's effective
  concentration of 55 M in pure water. This "pKa" is used to compare
  the instrinsic acidity of water to other weak acids. Water is an
  exceptionally weak acid or base.
  

___
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 2/7/2012 3:42 PM, Kevin Jin wrote:

  Oops, It should be: [H3O+]/[OH-]= 50/50

Kw = [H3O+][OH-],

pH = pKa +log ([OH-]/[H2O])

H3O+ concentration of pure water is 10^-7 mol/L

total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right?

Regards,

Kevin


On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu wrote:

  
Hi Kevin,

Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid.  The H30+ concentration of pure water is 10^-7 Molar.  In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+).  This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or  metal that protonates or deprotonates the water to 'activate it'.


Best regards,

Z


***
Zachary A. Wood, Ph.D.
Assistant Professor
Department of Biochemistry  Molecular Biology
University of Georgia
Life Sciences Building, Rm A426B
120 Green Street
Athens, GA  30602-7229
Office: 706-583-0304
Lab:    706-583-0303
FAX: 706-542-1738
***






On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote:



  As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
should be 50/50.
In this case, we may need to consider water in two formats:

H2O vs. H3O+

When we say water as acid, it usually stands for H3O+ in chemistry. In
chemical equation, H+ represents H3O+.

In enzyme catalysis, water as a general acid sounds reasonable under
pH 7.0. In some famous paper, water has been concluded as the general
base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
to be reconsidered.
.
Recently, I have read papers for pKa perturbation. I am also
interested in the general base of Asp and Glu in enzyme catalysis.


I will be very happy to read your paper in the future.

Regards,

Kevin Jin

On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote:

  
Dear colleagues,

We have solved the crystal structure of a human enzyme. The pKa of a
catalytically critical aspartic acid has increased to 6.44. It is hydrogen
bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
proton during the catalysis. Can anybody help me a) interpret the
significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
in context with the catalysis? Is this advantageous or detrimental? b) How
is pKa related to an amino acids’ ability to force a water molecule to
donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
similar increase in pKa values observed for aspartic acids before? I would
be grateful if anybody could explain or comment on the above queries.

Deepak Oswal

  
  




  

  



Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Horacio Botti
Dear all,

for further discussion

I believe that using the 0-14 pH scale assumes water activity of pure
water, something that is surely not matched in the surface or pocket of a
protein, so keeping this in mind I always prefer to speak about apparent
pKa of a group if talking about a non solvent-exposed group.

Horacio

2012/2/7 Roger Rowlett rrowl...@colgate.edu

  No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C.

 So at pH 7.0, you have 10^-7 M each at equilibrium no matter how you slice
 it or whatever else is in solution. If equilibrium [H3O+] goes up [OH-]
 goes down commensurately.

 The pKa of water as an acid is based on Kw and water's effective
 concentration of 55 M in pure water. This pKa is used to compare the
 instrinsic acidity of water to other weak acids. Water is an exceptionally
 weak acid or base.


 ___
 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 2/7/2012 3:42 PM, Kevin Jin wrote:

 Oops, It should be: [H3O+]/[OH-]= 50/50

 Kw = [H3O+][OH-],

 pH = pKa +log ([OH-]/[H2O])

 H3O+ concentration of pure water is 10^-7 mol/L

 total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right?

 Regards,

 Kevin


 On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu 
 z...@bmb.uga.edu wrote:

  Hi Kevin,

 Hate to point this out, but under pH 7.0, the protonation state of water is 
 not 50:50, and it is not a good acid.  The H30+ concentration of pure water 
 is 10^-7 Molar.  In pure water (assuming 55.5 M) only 1:555,000,000 water 
 molecules is in the protonated, charged state (H3O+).  This is why when an 
 enzyme uses water in its mechanism as a nucleophile, base, or acid, there is 
 usually an acid/base catalyst or  metal that protonates or deprotonates the 
 water to 'activate it'.


 Best regards,

 Z


 ***
 Zachary A. Wood, Ph.D.
 Assistant Professor
 Department of Biochemistry  Molecular Biology
 University of Georgia
 Life Sciences Building, Rm A426B
 120 Green Street
 Athens, GA  30602-7229
 Office: 706-583-0304
 Lab:706-583-0303
 FAX: 706-542-1738
 ***






 On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote:


  As we know, the pKa of water is 15.7. Under pH 7.0, its protonation
 should be 50/50.
 In this case, we may need to consider water in two formats:

 H2O vs. H3O+

 When we say water as acid, it usually stands for H3O+ in chemistry. In
 chemical equation, H+ represents H3O+.

 In enzyme catalysis, water as a general acid sounds reasonable under
 pH 7.0. In some famous paper, water has been concluded as the general
 base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a
 hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need
 to be reconsidered.
 .
 Recently, I have read papers for pKa perturbation. I am also
 interested in the general base of Asp and Glu in enzyme catalysis.


 I will be very happy to read your paper in the future.

 Regards,

 Kevin Jin

 On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com 
 deepos...@gmail.com wrote:

  Dear colleagues,

 We have solved the crystal structure of a human enzyme. The pKa of a
 catalytically critical aspartic acid has increased to 6.44. It is hydrogen
 bonded (2.8 Angstroms) to a water molecule that is supposed to donate a
 proton during the catalysis. Can anybody help me a) interpret the
 significance of this increase in pKa of the aspartic acid from 3.8 to 6.44
 in context with the catalysis? Is this advantageous or detrimental? b) How
 is pKa related to an amino acids’ ability to force a water molecule to
 donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated
 irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have
 similar increase in pKa values observed for aspartic acids before? I would
 be grateful if anybody could explain or comment on the above queries.

 Deepak Oswal




-- 
Horacio Botti
PhD MD
Protein Crystallography Unit
Institut Pasteur of Montevideo


Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Matthew Franklin

On 2/7/12 4:02 PM, Jacob Keller wrote:

Dear CCP4BB,

this is perhaps my most egregious off-topic post, but can anyone
explain why the following reference is not findable in PubMed? I can
get it from the ACS website, but not on PubMed or elsewhere. The
journal is on PubMed--is it perhaps because it's funded by ExxonMobil?
Very strange...

Jacob




Hi Jacob -

PubMed isn't universal - its goal is to cover the biomedical literature, 
so articles on the margins, like organic chemistry, materials science 
(even crystallography!) aren't fully covered.  If you look at PubMed's 
total coverage of the journal Macromolecules, you'll see that it's 
extremely patchy: lots of articles from 1977, then almost nothing until 
a whole fleet of articles in 1998, then almost nothing again until 
2007.  Your article must have fallen into one of these coverage holes.


Either that, or it's THE MAN suppressing the research needed to cure 
cancer and the common cold, and build a car that runs on water...


:)

- Matt

--
Matthew Franklin, Ph. D.
Senior Research Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(646) 275-7165


[ccp4bb] Generating parameters/cif files for macrocyclic ligands

2012-02-07 Thread Joel Tyndall
Hi folks,

I have an intriguing problem. I'm trying to generate a cif file for a 
macrocyclic peptide (of the likes in 
pdb1d4khttp://www.rcsb.org/pdb/explore/explore.do?structureId=1d4k). They are 
cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used 
PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is 
cyclic one cannot adjust the dihedral angles. I have previously done this using 
CNS where you can break the tricyclic peptide into residues and generate 
parameters to specify bonds/links between the residues (which allows this kind 
of movement). I can't come up with a way to do this  without using CNS. I have 
looked ta J-ligand which allows for one link between two separate residues 
which precludes a macrocycle. I have looked at sketcher within CCP4 which reads 
the pdb files but I don't believe this can be done here. Within Coot I can 
refine the whole ligand but not certain components.

Any suggestions greatly appreciated . ( I may stick to coot refinement with 
fixed atoms at this stage)

Regards

Joel

_
Joel Tyndall, PhD

Senior Lecturer in Medicinal Chemistry
National School of Pharmacy
University of Otago
PO Box 56 Dunedin 9054
New Zealand
Skype: jtyndall
http://www.researcherid.com/rid/C-2803-2008
Pukeka Matua
Te Kura Taiwhanga Putaiao
Te Whare Wananga o Otago
Pouaka Poutapeta 56 Otepoti 9054
Aotearoa

Ph / Waea   +64 3 4797293
Fax / Waeawhakaahua +64 3 4797034



Re: [ccp4bb] No diffraction

2012-02-07 Thread Jesse
This might be obvious, but make sure you washed the crystals
thoroughly before dissolving them for SDS-PAGE or mass spec

-Jesse

On Thu, Jan 26, 2012 at 10:48 AM, Katherine Sippel
katherine.sip...@gmail.com wrote:
 Might I suggest consulting the CCP4 user community wiki on the topic:

 http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality

 Good luck,

 Katherine



 On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu theresah...@live.com
 wrote:

 Dear crystallographers

 I have a protein of 90 kDa forming dimers. Crystals formed with microbatch
 and vapor diffusion method in 24 hours but no diffraction at home source.
 Dissolved crystals was confirmed to be the protein with mass spec.

 Any suggestions to improve diffraction would be welcome.

 Thanking you in advance.

 Theresa




Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Jacob Keller
Well, perhaps it is because size exclusion chromatography is used so
little in the life sciences؟, and who really cares how it works
anyway?؟

JPK


On Tue, Feb 7, 2012 at 3:18 PM, Matthew Franklin mfrank...@nysbc.org wrote:
 On 2/7/12 4:02 PM, Jacob Keller wrote:

 Dear CCP4BB,

 this is perhaps my most egregious off-topic post, but can anyone
 explain why the following reference is not findable in PubMed? I can
 get it from the ACS website, but not on PubMed or elsewhere. The
 journal is on PubMed--is it perhaps because it's funded by ExxonMobil?
 Very strange...

 Jacob



 Hi Jacob -

 PubMed isn't universal - its goal is to cover the biomedical literature, so
 articles on the margins, like organic chemistry, materials science (even
 crystallography!) aren't fully covered.  If you look at PubMed's total
 coverage of the journal Macromolecules, you'll see that it's extremely
 patchy: lots of articles from 1977, then almost nothing until a whole fleet
 of articles in 1998, then almost nothing again until 2007.  Your article
 must have fallen into one of these coverage holes.

 Either that, or it's THE MAN suppressing the research needed to cure cancer
 and the common cold, and build a car that runs on water...

 :)

 - Matt

 --
 Matthew Franklin, Ph. D.
 Senior Research Scientist
 New York Structural Biology Center
 89 Convent Avenue, New York, NY 10027
 (646) 275-7165





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] SAXS workshop

2012-02-07 Thread Richard Gillilan
The MacCHESS BioSAXS Essentials minicourse filled up just a few days after we 
made the announcement over a week ago. Hopefully the overflow will find their 
way to Brookhaven and other places. To date, there has NOT been a lot of 
interest among potential organizers in holding such a course at the annual 
American Crystallographic Association meeting. 

Given the intense interest in the subject, I would like to know if any of you 
CCP4 readers would be interested in attending a lecture-only BioSAXS HOWTO 
workshop at the ACA meeting in Hawaii 2013. Please be realistic, I know many 
would LIKE to go ... but would you actually be able to go? I would also be 
interested in hearing from any interested potential organizers.

(Please also keep in mind that SAS2012 is in Australia Nov 2012, so that may 
make a difference in your travel plans). 

Richard Gillilan
chair-elect ACA SAXS SIG
MacCHESS
Cornell University
Ithaca, NY




On Feb 6, 2012, at 1:06 PM, Robert Sweet wrote:

 And here's one from Brookhaven -- app. deadline is three weeks away:
 
 http://workshops.ps.bnl.gov/default.aspx?w=SAXSMar2012
 
 http://www.chess.cornell.edu/BioSAXS%20course/index.htm
 
 Getting Started in Biological Small-Angle X-ray Solution Scattering
 Feb 24-26, 2012
 Students will have the opportunity to collect data on CHESS beamlines using 
 protein
 standards and/or their own samples.
 
 
 -- 
 =
Robert M. Sweet E-Dress: sw...@bnl.gov
Group Leader, PXRR: Macromolecular   ^ (that's L
  Crystallography Research Resource at NSLSnot 1)
  http://px.nsls.bnl.gov/
Biology Dept
Brookhaven Nat'l Lab.   Phones:
Upton, NY  11973631 344 3401  (Office)
U.S.A.  631 344 2741  (Facsimile)
 =


[ccp4bb] shape complementarity

2012-02-07 Thread Francois Berenger

Hello,

After following the discussion on
[ccp4bb] shape complementarity between protein and DNA surface,
is there someone here able to explain simply what the SC software
of CCP4 is calculating?

I mean, is there some intuitive/easy to understand explanation of what 
SC is calculating?


I know I should read the corresponding paper, but I'd like
someone to enlighten me before so I have better chances of understanding 
the article.


Thanks,
Francois.


Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread John Newitt
This looks like one of those journals not routinely indexed by the NLM. There 
are some issues in Medline, but not all. I only found one article from 2004.

http://www.ncbi.nlm.nih.gov/nlmcatalog/365316

John

Sent from my iPad

On Feb 7, 2012, at 4:02 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote:

 Dear CCP4BB,
 
 this is perhaps my most egregious off-topic post, but can anyone
 explain why the following reference is not findable in PubMed? I can
 get it from the ACS website, but not on PubMed or elsewhere. The
 journal is on PubMed--is it perhaps because it's funded by ExxonMobil?
 Very strange...
 
 Jacob
 
 
 Article
 A Study of the Separation Principle in Size Exclusion Chromatography
 AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240
 KB]FiguresCiting Articles
 Thomas Sun*
 Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr.,
 Baytown, Texas 77520-2101
 Ronald R. Chance, William W. Graessley,† and David J. Lohse
 Corporate Strategic Research, ExxonMobil Research and Engineering,
 Annandale, New Jersey 08801
 Macromolecules, 2004, 37 (11), pp 4304–4312
 DOI: 10.1021/ma030586k
 Publication Date (Web): April 29, 2004
 Copyright © 2004 American Chemical Society
 
 -- 
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***


Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Francis E Reyes
I'm going with Jurgen on this one. 

http://img27.imageshack.us/img27/3232/pastedgraphic1.png


Sad was the day when I mounted this puppy and it shot to 8-10A. Room 
temperature. And messing around with cryos didn't help either. 


Can't remember the size, but I think I had scooped it with a 0.8 mm loop.  

I should've mounted it on a ring and given it to my wife. And that's not 
chromatic artifact, the ligand was red. 

On a side note, I had a very small crystal embedded in a chunk of ice at the 
end of a 0.025 mm loop.  Couldn't even see it on the very nice on-axis cameras 
at the ALS. I shot blindly into the ice.. .it diffracted to about 1.8A (and the 
ice wasn't bad at all)


F


On Feb 7, 2012, at 9:52 AM, Bosch, Juergen wrote:

 Hi Enrico,
 
 I was just looking at non-optimal cryo-conditions and the original posters 
 starting point.
 Of course if you have a good cryo bigger is better for the reasons you write 
 but if you have no clue how your crystals will perform then I'd rather go for 
 small to be cautious and also have those around and not only the big ones 
 which everybody mounts because they looks so nice. To be disappointed by big 
 crystals is often not a surprise to me and if you have not tried small 
 crystals from the same batch well then you missed 50% of your chances to 
 solve s structure with the first light the crystals saw.
 
 Jürgen
 
 

-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder


Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jacob Keller
 Sad was the day when I mounted this puppy and it shot to 8-10A. Room 
 temperature. And messing around with cryos didn't help either.

But if that puppy had been smaller, it might have diffracted even
worse, and just think if that little icy crystal had been bigger...

JPK




 Can't remember the size, but I think I had scooped it with a 0.8 mm loop.

 I should've mounted it on a ring and given it to my wife. And that's not 
 chromatic artifact, the ligand was red.

 On a side note, I had a very small crystal embedded in a chunk of ice at the 
 end of a 0.025 mm loop.  Couldn't even see it on the very nice on-axis 
 cameras at the ALS. I shot blindly into the ice.. .it diffracted to about 
 1.8A (and the ice wasn't bad at all)


 F


 On Feb 7, 2012, at 9:52 AM, Bosch, Juergen wrote:

 Hi Enrico,

 I was just looking at non-optimal cryo-conditions and the original posters 
 starting point.
 Of course if you have a good cryo bigger is better for the reasons you write 
 but if you have no clue how your crystals will perform then I'd rather go 
 for small to be cautious and also have those around and not only the big 
 ones which everybody mounts because they looks so nice. To be disappointed 
 by big crystals is often not a surprise to me and if you have not tried 
 small crystals from the same batch well then you missed 50% of your chances 
 to solve s structure with the first light the crystals saw.

 Jürgen



 -
 Francis E. Reyes M.Sc.
 215 UCB
 University of Colorado at Boulder



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] 2

2012-02-07 Thread Krystle Williams McLaughlin
...a weight is lifted off my shoulders  
http://www.flipperkiste.de/work.link.php?jgyahooID=65l2
  

Re: [ccp4bb] shape complementarity

2012-02-07 Thread Mike Lawrence
Hi Francois

Here's a one-liner. The major concept behind the Sc coefficient is that it 
measures the extent to which, on average, the normal vectors between 
closest-neighbour opposing points within the molecular interface are 
antiparallel. 

Sc=1 implies that the surfaces fit exactly, all such vectors are perfectly 
antiparallel. Heuristically, Sc values of 0.86 are about as good as 
protein-protein interfaces get (see Nature. 2005 435, pp773-8). Values below 
0.65 indicate relatively poor shape complementarity.

Use of a normal vector -based metric is considered superior to a distance-based 
metric, though Sc does have a distance-based weight applied to the normal dot 
products. Critical to this calculation is that the boundary of the buried 
molecular interface has to be discarded from the measure, as this region is 
intrinsically geometrically divergent. Sc is thus computed across only that 
part of the buried surface that might be expected to be shape complementarity, 
which makes it somewhat ill-suited to smaller interfaces.

All these details are in the JMB paper, which, unfortunately, there is no 
substitute for reading :-)

sincerely

Mike

Mike Lawrence, PhD

Associate Professor and WEHI Fellow
Division of Structural Biology
Walter and Eliza Hall Institute of Medical Research
1G Royal Parade, Parkville
Victoria 3052, AUSTRALIA

Tel. 61-3-9345-2693   
Fax 61-3-9345-2686
Email: lawre...@wehi.edu.au




On 08/02/2012, at 12:08 PM, Francois Berenger wrote:

 Hello,
 
 After following the discussion on
 [ccp4bb] shape complementarity between protein and DNA surface,
 is there someone here able to explain simply what the SC software
 of CCP4 is calculating?
 
 I mean, is there some intuitive/easy to understand explanation of what SC is 
 calculating?
 
 I know I should read the corresponding paper, but I'd like
 someone to enlighten me before so I have better chances of understanding the 
 article.
 
 Thanks,
 Francois.



__
The information in this email is confidential and intended solely for the 
addressee.
You must not disclose, forward, print or use it without the permission of the 
sender.
__


Re: [ccp4bb] shape complementarity

2012-02-07 Thread Francois Berenger

On 02/08/2012 12:47 PM, Mike Lawrence wrote:

Hi Francois

Here's a one-liner. The major concept behind the Sc coefficient is that it measures the 
extent to which, on average, the normal vectors between closest-neighbour 
opposing points within the molecular interface are antiparallel.

Sc=1 implies that the surfaces fit exactly, all such vectors are perfectly 
antiparallel. Heuristically, Sc values of 0.86 are about as good as 
protein-protein interfaces get (see Nature. 2005 435, pp773-8). Values below 
0.65 indicate relatively poor shape complementarity.

Use of a normal vector -based metric is considered superior to a distance-based 
metric, though Sc does have a distance-based weight applied to the normal dot 
products. Critical to this calculation is that the boundary of the buried 
molecular interface has to be discarded from the measure, as this region is 
intrinsically geometrically divergent. Sc is thus computed across only that 
part of the buried surface that might be expected to be shape complementarity, 
which makes it somewhat ill-suited to smaller interfaces.

All these details are in the JMB paper, which, unfortunately, there is no 
substitute for reading :-)


Thanks a lot for this very nice answer.

I will definitely read your JMB article.

Best regards,
Francois.


[ccp4bb] My hotmail address was hacked!

2012-02-07 Thread Krystle Williams McLaughlin

Hi all,Please don't click on any links in emails from me, my hotmail password 
was compromised and it emailed my entire address book malicious links.Hope 
everyone is well!Krystle 
-- 
Krystle J. McLaughlin, Ph.D.

SPIRE Postdoctoral Fellow

Redinbo Group, Department of Chemistry

University of North Carolina at Chapel Hill



  

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Edward A. Berry

Bosch, Juergen wrote:

Hi Dirk,

I remember a neat paper don't recall who wrote it. I think it was in Acta D 
where the
authors made a tiny probe the size of an elongated crystal glued to a 
[/Advertisement on]
Hampton loop [/Advertisement off]. The probe was a temperature sensor and they 
recorded
the cooling rate under different methods. The winner as far as I recall was 
freezing in
liquid propane for the lack of the missing gas layer, but the second best 
method was LN2.
Propane for whatever reason has gone extinct in certain areas of the world :-) 
. I'll try
to find that reference but perhaps somebody else on this highly educated board 
knows which
paper I'm referring to. I want to say it was published around 2004-2006.


Not highly educated, but I remember hearing Haken Hope talk about
this experiment at a workshop at SSRL- cooling a thermocouple or
thermister in the cold stream vs in LN2.
Maybe described here:
Cryocrystallography of biological macromolecules: a generally applicable method.
Hope H. Acta Crystallogr B. 1988 Feb 1;44 ( Pt 1):22-6.

BTW I don't thing the greater cooling rate with ethane/propane is due
to greater heat capacity so much as the fact that LN2 is used at
it's boiling point: heat capacity is irrelevant since it can't absorb
any more heat as a liquid, latent heat of vaporization is the reelevant
parameter, but once it is vaporized the gas has low heat capacity and
thermal conductivity.
The liquid hydrocarbons are prepared by chilling to around their
freezing point (hydrocarbon slush) so can absorb a lot of heat before any gas 
forms.



Jürgen

On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote:


Dear Jürgen,

Am 07.02.12 16:58, schrieb Bosch, Juergen:
snip

Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2
leads to a quicker freeze of your material.

/snip

Are you sure? There was a publication by Warkentin et al. [1] about a
cold gas layer above liquid nitrogen that reduces the expected cooling
rate a lot!
My very personal experience is, that cryo-cooling in the N2-stream
worked better for me than in LN2 in a variety of projects - but the
reason could just be me ;-)

Best regards,

Dirk.

[1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert
E Thorne: Hyperquenching for protein cryocrystallography, J. Appl.
Crystallogr., 39, 805-811 (2006)

--

***
Dirk Kostrewa
Gene Center Munich
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail:kostr...@genzentrum.lmu.de mailto:kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://web.mac.com/bosch_lab/






[ccp4bb] pH optimisation for crystallisation

2012-02-07 Thread sreetama das
Dear all,
   I have a 17 KDa protein that gives crystals in a condition that has 
0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not 
diffract. I have tried additives, but they haven't improved the crystals. I 
intend to vary the pH of the condition.
   My questions are-
1. should the buffer be kept the same or can it also be changed (as long as the 
desired pH is within the range of both the buffers)?
2. in case of a different buffer, should its molarity be the same as that of 
the original one in the crystallization condition?

regards,
sreetama


Re: [ccp4bb] pH optimisation for crystallisation

2012-02-07 Thread VAN RAAIJ , MARK JOHAN

Dear Sreetama,
First of all, there are no hard-and-fast rules for successful  
crystallisation, try changing as many different variables as possible  
and go with what works.
Having said that, yes, next I would go for a grid optimisation varying  
the pH in 0.2 or 0.5 units over as wide a range as the buffer can  
reasonable tolerate at the same molarity, and try different  
precipitant concentrations on the other axis.
If you have enough protein, try plates at different temperatures as  
well, and different protein concentrations (in multidrop wells you can  
do this in the same experiment).
A very important variable is the protein preparation itself, prepare  
more protein regularly and try to improve on purity and concentration.

Mark

Quoting sreetama das:


Dear all,
   I have a 17 KDa protein that gives crystals in a  
condition that has 0.1M bis-tris pH 6.5. The crystals are thin  
needle clusters and do not diffract. I have tried additives, but  
they haven't improved the crystals. I intend to vary the pH of the  
condition.

   My questions are-
1. should the buffer be kept the same or can it also be changed (as  
long as the desired pH is within the range of both the buffers)?
2. in case of a different buffer, should its molarity be the same as  
that of the original one in the crystallization condition?


regards,
sreetama





Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es