Re: [ccp4bb] Active aggregates?

[James Stroud]

Just try crystallizing it. What is a crystal but a "massive  
aggregate"? That they are still soluble and active is great news.


As a grad student, I had a similar phenomenon with an early project. I  
showed a gel in group meeting where both activity and aggregation were  
obvious, said the aggregate was no problem, got ridiculed when I said  
I was going to throw it in trays despite what anyone said, had giant  
crystals after a few trays, and solved the structure with miras.


Get the structure and then worry about why it's aggregating. The  
structure will probably provide you with the clues you need.


On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote:



Dear all,

This is a question on how to cope with the protein that seems to form
massive aggregates in solution but enzymatically active.

I'm working on a protein whose molecular weight is around 70kDa and  
can be
divided into two domains (say A and B domains). We expressed this  
protein in

E.coli fused with GST and purified using some chromatography. The GST
affinity chromatography works well and proteinase digestion to  
remove the

tag does wonders too. The purified protein was confirmed to be active
enough, we can detect both activities from these two domains. But the
retention time from the gel filtration clearly shows it is awfully
aggregated (comes out at the void region). DLS measurement indicates  
the

averaged diameter is around 45 nm, which I feel is a bit too long.
Analytical ultracentrifuge result implies that the distribution of the
molecular species is wide, some portion got precipitated with 1K rcf  
(means
the molecular weight is more than 5MDa) and the rest is ranging from  
1MD to

5MDa with a peak at 1MDa.
I made new two constructs covering the A and B domains respectively,  
both of
them are active again, but only the A domain has got the same  
symptom as the

intact protein. The B domain seems to exist as a monomer in solution.

Here come my questions, (I) How can I interpret this phenomenon?  
(II) Is
there anything we can try to change the situation? (III) Does it  
make sense
to try crystallization? (probably not).(IV) Has anyone got such  
experience?


I tried the methylation on lysine side chains, I also tried the  
buffer with
0.2M arginine or 10% glycerol but the all the results just seem  
hopeless.

The protein before the proteinase treatment also comes out at the void
region from the gel filtration.


cheers

toyoyuki

--?
Toyoyuki Ose
o...@sci.hokudai.ac.jp

Graduate School of Life Science
Hokkaido University
N21W11 Kita-ku, Sapporo
001-0021 Japan

Re: [ccp4bb] suggestions for SIR with strong diffraction anisotropy in the derivative

[Artem Evdokimov]

Well, this is of course just my undereducated two cents but the first
question I'd ask is: asuming that dataset #2 is a real derivative - can I
find realistic sites? What are you basing your resolution cutoff on - in the
diffraction images, do you visually observe anisotropy? 

It's not uncommon for derivatives to change diffraction properties of the
crystal anisotropically - for instance if a derivative site happens to be on
a major crystal contact in a specific direction within the crystal.

Can you get better data (better crystals)? It's awfully hard to trace
experimental maps at 4+ A especially if you have no NCS multiples to help
you with crummy phases.

Artem

"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone" 
 Jorge Luis Borges
 

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Francis E Reyes
Sent: Wednesday, August 26, 2009 4:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for SIR with strong diffraction anisotropy in
the derivative

Hi all

I'm trying SIR on my crystal both are C222.


Native
Cell Dimensions: 97.66 243.45 87.62 90 90 90
Resolution: 4.3

Derivative
Cell Dimensions: 96.9100 244.66 87.61 90 90 90
Resolution: 4.7

.  I plan to scale together using FHScal. Ctruncate says that my  
derivative has strong diffraction anisotropy. How concerned should I  
be about this at the level of phasing? Is there anything to be done  
with the anisotropy issue?

Thanks


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

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] Active aggregates?

[Artem Evdokimov]

Dear Ose,

This is unfortunately a fairly common issue. There are multiple ways to
rationalize what's happening - and even more approaches to reducing the
aggregation issues. It would take too long to list all the options here -
however I would look at the earliest stages of your expression and
purification and try to determine if aggregation is the 'inevitable outcome'
of inherent properties of your protein - or is it the outcome of inadequate
expression, handling during lysis, purification, etc.

Beyond the abovementioned considerations, the following may be useful to
think about: 

a) is the specific activity of your protein close to maximum attainable (or
at least biologically reasonable) value?
b) if you can separate aggregates away from monomers (or proper oligomers) -
does the protein re-aggregate with time or upon concentration?
c) does aggregation state of your protein change with time or during the
course of purification?
d) what is the effect of ionic strength, pH, nature of buffer components,
additives, ligands (substrates?), and so forth?

(a) may be difficult to answer unless you have literature or unpublished
data on this enzyme or its close relatives
(b) is one of the earliest experiments I'd try in the absence of all other
knowledge
(c) should be tracked as you work with the protein as soon as you suspect
that there may be issues
(d) is probably the second experiment I'd try w/o any other knowledge. If
you can use Thermofluor or some other high-throughput method to analyze the
behavior of your protein exposed to various conditions, then you can screen
quite an array of options very quickly

Good luck,

Artem

"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone" 
 Jorge Luis Borges
 
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ose
toyoyuki
Sent: Wednesday, August 26, 2009 7:55 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Active aggregates?

 
Dear all,
 
This is a question on how to cope with the protein that seems to form
massive aggregates in solution but enzymatically active.
 
I'm working on a protein whose molecular weight is around 70kDa and can be
divided into two domains (say A and B domains). We expressed this protein in
E.coli fused with GST and purified using some chromatography. The GST
affinity chromatography works well and proteinase digestion to remove the
tag does wonders too. The purified protein was confirmed to be active
enough, we can detect both activities from these two domains. But the
retention time from the gel filtration clearly shows it is awfully
aggregated (comes out at the void region). DLS measurement indicates the
averaged diameter is around 45 nm, which I feel is a bit too long.
Analytical ultracentrifuge result implies that the distribution of the
molecular species is wide, some portion got precipitated with 1K rcf (means
the molecular weight is more than 5MDa) and the rest is ranging from 1MD to
5MDa with a peak at 1MDa.
I made new two constructs covering the A and B domains respectively, both of
them are active again, but only the A domain has got the same symptom as the
intact protein. The B domain seems to exist as a monomer in solution.
 
Here come my questions, (I) How can I interpret this phenomenon? (II) Is
there anything we can try to change the situation? (III) Does it make sense
to try crystallization? (probably not).(IV) Has anyone got such experience?
 
I tried the methylation on lysine side chains, I also tried the buffer with
0.2M arginine or 10% glycerol but the all the results just seem hopeless.
The protein before the proteinase treatment also comes out at the void
region from the gel filtration.
 
 
cheers

toyoyuki

--?
Toyoyuki Ose
o...@sci.hokudai.ac.jp

Graduate School of Life Science
Hokkaido University
N21W11 Kita-ku, Sapporo
001-0021 Japan

Re: [ccp4bb] Active aggregates?

[John Tanner]

You could try adding detergents.  We had a case a few years ago of an enzyme
that was highly active but also highly aggregated. Addition of n-octyl
beta-d-glucopyranoside significantly lowered the degree of aggregation such
that crystals could be grown.  Lowering the protein concentration also
helped. The work is described in an Acta F paper:

Cloning, purification and crystallization of Thermus thermophilus proline
dehydrogenase.

White TA, Tanner JJ.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Aug 1;61(Pt 8):737-9.

Jack


-- 
John J. Tanner
Professor of Chemistry and Biochemistry
University of Missouri-Columbia
125 Chemistry Building
Columbia, MO 65211
Phone: 573-884-1280
Fax: 573-882-2754
Email: tanne...@missouri.edu
http://www.chem.missouri.edu/TannerGroup/tanner.html





On 8/26/09 7:55 PM, "ose toyoyuki"  wrote:

> 
>  
> Dear all,
>  
> This is a question on how to cope with the protein that seems to form
> massive aggregates in solution but enzymatically active.
>  
> I'm working on a protein whose molecular weight is around 70kDa and can be
> divided into two domains (say A and B domains). We expressed this protein in
> E.coli fused with GST and purified using some chromatography. The GST
> affinity chromatography works well and proteinase digestion to remove the
> tag does wonders too. The purified protein was confirmed to be active
> enough, we can detect both activities from these two domains. But the
> retention time from the gel filtration clearly shows it is awfully
> aggregated (comes out at the void region). DLS measurement indicates the
> averaged diameter is around 45 nm, which I feel is a bit too long.
> Analytical ultracentrifuge result implies that the distribution of the
> molecular species is wide, some portion got precipitated with 1K rcf (means
> the molecular weight is more than 5MDa) and the rest is ranging from 1MD to
> 5MDa with a peak at 1MDa.
> I made new two constructs covering the A and B domains respectively, both of
> them are active again, but only the A domain has got the same symptom as the
> intact protein. The B domain seems to exist as a monomer in solution.
>  
> Here come my questions, (I) How can I interpret this phenomenon? (II) Is
> there anything we can try to change the situation? (III) Does it make sense
> to try crystallization? (probably not).(IV) Has anyone got such experience?
>  
> I tried the methylation on lysine side chains, I also tried the buffer with
> 0.2M arginine or 10% glycerol but the all the results just seem hopeless.
> The protein before the proteinase treatment also comes out at the void
> region from the gel filtration.
>  
>  
> cheers
> 
> toyoyuki
> 
> --?
> Toyoyuki Ose
> o...@sci.hokudai.ac.jp
> 
> Graduate School of Life Science
> Hokkaido University
> N21W11 Kita-ku, Sapporo
> 001-0021 Japan

[ccp4bb] Active aggregates?

[ose toyoyuki]

 
Dear all,
 
This is a question on how to cope with the protein that seems to form
massive aggregates in solution but enzymatically active.
 
I'm working on a protein whose molecular weight is around 70kDa and can be
divided into two domains (say A and B domains). We expressed this protein in
E.coli fused with GST and purified using some chromatography. The GST
affinity chromatography works well and proteinase digestion to remove the
tag does wonders too. The purified protein was confirmed to be active
enough, we can detect both activities from these two domains. But the
retention time from the gel filtration clearly shows it is awfully
aggregated (comes out at the void region). DLS measurement indicates the
averaged diameter is around 45 nm, which I feel is a bit too long.
Analytical ultracentrifuge result implies that the distribution of the
molecular species is wide, some portion got precipitated with 1K rcf (means
the molecular weight is more than 5MDa) and the rest is ranging from 1MD to
5MDa with a peak at 1MDa.
I made new two constructs covering the A and B domains respectively, both of
them are active again, but only the A domain has got the same symptom as the
intact protein. The B domain seems to exist as a monomer in solution.
 
Here come my questions, (I) How can I interpret this phenomenon? (II) Is
there anything we can try to change the situation? (III) Does it make sense
to try crystallization? (probably not).(IV) Has anyone got such experience?
 
I tried the methylation on lysine side chains, I also tried the buffer with
0.2M arginine or 10% glycerol but the all the results just seem hopeless.
The protein before the proteinase treatment also comes out at the void
region from the gel filtration.
 
 
cheers

toyoyuki

--?
Toyoyuki Ose
o...@sci.hokudai.ac.jp

Graduate School of Life Science
Hokkaido University
N21W11 Kita-ku, Sapporo
001-0021 Japan

[ccp4bb] how to recreate missing import.def file

[Beth A Wurzburg]

Hi All, 

After transferring a lot of files from one computer to another, we
discovered a problem when we launched ccp4i.  The project database
job list is empty even though all the .log files, etc. are clearly
present.  We checked the CCP4_DATABASE directory and discovered that
the import.def file didn't make it over (and it's hard to recover
at the moment).  Is there an easy way to recreate it?  The other
.def files made it over and we can see the log files, etc.


  Thanks,


 Beth

[ccp4bb] MrBUMP

[riya doreen]

Hi everyone,

I am trying to run MrBUMP through the CCP4 6.1.1 but the program is
complaining that no multiple alignment programs like mafft, clustalw,
clustalw2, probcons, or t_coffee were found on the system.

Will I need to install all these programs and will they need to be in the
CCP4 directory ?
Thanks

[ccp4bb] suggestions for SIR with strong diffraction anisotropy in the derivative

[Francis E Reyes]

Hi all

I'm trying SIR on my crystal both are C222.


Native
Cell Dimensions: 97.66 243.45 87.62 90 90 90
Resolution: 4.3

Derivative
Cell Dimensions: 96.9100 244.66 87.61 90 90 90
Resolution: 4.7

.  I plan to scale together using FHScal. Ctruncate says that my  
derivative has strong diffraction anisotropy. How concerned should I  
be about this at the level of phasing? Is there anything to be done  
with the anisotropy issue?


Thanks


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

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

[ccp4bb] Arpwarp not recognised by latest version of ccp4

[Sylvia Fanucchi]

  

Hi

Does anyone know how to configure ArpWARP manually so that it will be
recognized by the latest version of ccp4?

 

Best regards

Sylvia Fanucchi Ph.D

Protein Structure-Function Research Unit
East Campus, Gate House Room 416
School of Molecular and Cell Biology
University of the Witwatersrand
Johannesburg 2050
South Africa

Tel: +27 (11) 717-6348 Fax: +27 (11) 717-6351 
E-mail: sylvia.fanuc...@wits.ac.za   

 


This communication 
is intended for the addressee only. It is confidential. If you have received 
this communication in error, please notify us immediately and destroy the 
original message. You may not copy or disseminate this communication without 
the permission of the University. Only authorized signatories are competent to 
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advised that the content of this message may not be legally binding on the 
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are not necessarily the views and opinions of The University of the 
Witwatersrand, Johannesburg. All agreements between the University and 
outsiders are subject to South African Law unless the University agrees in 
writing to the contrary.
<>

Re: [ccp4bb] packing diagrammes

[Phoebe Rice]

I'd use MAMA to make an extremely simplified mask (either
based on a couple of nicely-positioned spheres, or excessively
smoothed) and they display it with its symmetry mates.

 Original message 
>Date: Wed, 26 Aug 2009 11:48:57 +0200
>From: Anita Lewit-Bentley   
>Subject: [ccp4bb] packing diagrammes  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear all,
>   does anyone know of a programme that would allow the
>   graphical representation of the packing of protein
>   molecules in a crystal? Ideally one would like to
>   represent the protein as an elipsoid (or similar
>   relatively simple shape) corresponding to its
>   volume, over a few unit cells so as to visualise the
>   continuity of solvent channels etc.
>   Thanks for any leads!
>   Anita
>
>   Anita Lewit-Bentley
>
>   Unité d'Immunologie Structurale
>   CNRS URA 2185
>   Département de Biologie Structurale & Chimie
>   Institut Pasteur
>   25 rue du Dr. Roux
>   75724 Paris cedex 15
>   FRANCE
>   Tel: 33- (0)1 45 68 88 95
>   FAX: 33-(0)1 40 61 30 74
>   email: ale...@pasteur.fr
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them 
  both in one book
Please do take a 
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] ACA Twinning Workshop talks now available

[George M. Sheldrick]

The PDF files of the talks from the workshop on macromolecular
twinning at the 2009 ACA Meeting in Toronto may now be dowloaded 
free from the ACA web site: http://www.amercrystalassn.org 

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582

Re: [ccp4bb] packing diagrammes

[Jack Tanner]

The cns script neighbours.inp might be useful for this purpose.



From: AntonioLeung 
Reply-To: AntonioLeung 
Date: Wed, 26 Aug 2009 21:46:26 +0800
To: 
Subject: Re: [ccp4bb] packing diagrammes

Hi, 
 
Pymol may do this work, use"Action-->generate-->symmetry mates"
 
 
-- Original --
From:  "Anita Lewit-Bentley";
Date:  Wed, Aug 26, 2009 05:48 PM
To:  "CCP4BB";
Subject:  [ccp4bb] packing diagrammes
 
Dear all, 

does anyone know of a programme that would allow the graphical
representation of the packing of protein molecules in a crystal? Ideally one
would like to represent the protein as an elipsoid (or similar relatively
simple shape) corresponding to its volume, over a few unit cells so as to
visualise the continuity of solvent channels etc.

Thanks for any leads!

Anita

Anita Lewit-Bentley

Unité d'Immunologie Structurale
CNRS URA 2185
Département de Biologie Structurale & Chimie
Institut Pasteur
25 rue du Dr. Roux
75724 Paris cedex 15
FRANCE

Tel: 33- (0)1 45 68 88 95
FAX: 33-(0)1 40 61 30 74
email: ale...@pasteur.fr

Re: [ccp4bb] packing diagrammes

[AntonioLeung]

Hi, 
  
 Pymol may do this work, use"Action-->generate-->symmetry mates"
   
  
  -- Original --
  From:  "Anita Lewit-Bentley";
 Date:  Wed, Aug 26, 2009 05:48 PM
 To:  "CCP4BB"; 
 
 Subject:  [ccp4bb] packing diagrammes

  
Dear all, 

 does anyone know of a programme that would allow the graphical representation 
of the packing of protein molecules in a crystal? Ideally one would like to 
represent the protein as an elipsoid (or similar relatively simple shape) 
corresponding to its volume, over a few unit cells so as to visualise the 
continuity of solvent channels etc.
 

 Thanks for any leads!
 

 Anita
 

 
Anita Lewit-Bentley
 

 Unité d'Immunologie Structurale
 CNRS URA 2185
 Département de Biologie Structurale & Chimie
 Institut Pasteur
 25 rue du Dr. Roux
 75724 Paris cedex 15
 FRANCE
 

 Tel: 33- (0)1 45 68 88 95
 FAX: 33-(0)1 40 61 30 74
 email: ale...@pasteur.fr

[ccp4bb] packing diagrammes

[Anita Lewit-Bentley]

Dear all,

does anyone know of a programme that would allow the graphical  
representation of the packing of protein molecules in a crystal?  
Ideally one would like to represent the protein as an elipsoid (or  
similar relatively simple shape) corresponding to its volume, over a  
few unit cells so as to visualise the continuity of solvent channels  
etc.


Thanks for any leads!

Anita

Anita Lewit-Bentley
Unité d'Immunologie Structurale
CNRS URA 2185
Département de Biologie Structurale & Chimie
Institut Pasteur
25 rue du Dr. Roux
75724 Paris cedex 15
FRANCE

Tel: 33- (0)1 45 68 88 95
FAX: 33-(0)1 40 61 30 74
email: ale...@pasteur.fr