Re: [ccp4bb] homology modeling

[Chen, Yu Wai]

I would recommend Modeller
http://www.salilab.org/modeller/

Wai
Suppose I have two proteins A and B, they are structurally homologous, 
however the sequence identity is only about 20%. A has crystal 
structure available, so if I want to model protein B, what's the best 
way to do it? I don't think I can just thread the sequence of B to A 
structure because the number of residues are different and there might 
be gaps and insertions in the alignment. Any suggestions of good programs?





--
Yu Wai Chen, PhDLecturer
King's College London, Randall Division +44-207-848-8206
New Hunt's House, Guy's Campus, London SE1 1UL, U.K.

Re: [ccp4bb] homology modeling

[Francisco J. Enguita]

Hi Rui

Very easy to use is EsyPred3D :

http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/

Also good performance server is 3d-JIGSAW :

http://bmm.cancerresearchuk.org/~3djigsaw/

Hope it helps

Cheers

Francisco

> Hi, All
> Suppose I have two proteins A and B, they are structurally homologous,
> however the sequence identity is only about 20%. A has crystal structure
> available, so if I want to model protein B, what's the best way to do it?
> I
> don't think I can just thread the sequence of B to A structure because the
> number of residues are different and there might be gaps and insertions in
> the alignment. Any suggestions of good programs?
>
> Thanks.
>
> Rui
>


-- 
-
Francisco J. Enguita, Ph.D.
Host-pathogen Interactions Group
Macromolecular Crystallography Laboratory
ITQB
EAN, Av. da República
2781-901 Oeiras
Portugal
Phone : +351-21-4469663
Fax : +351-21-4433644
E-mail : fengu...@itqb.unl.pt
-

Re: [ccp4bb] homology modeling

[Anastassis Perrakis]

Modeller is indeed an excellent program (so are some others) but,  
without being an expert, I was told some time ago and I am still under  
the impression, that homology modeling with 20% identity (and many  
insertions and deletions as you point out) is a dark art. Being able  
to suggest the identical fold with some threading algorithm, is likely  
all you will be able to do. A homology based model might be too much  
to ask.


A.

On May 5, 2009, at 11:39, Chen, Yu Wai wrote:


I would recommend Modeller
http://www.salilab.org/modeller/

Wai
Suppose I have two proteins A and B, they are structurally  
homologous,

however the sequence identity is only about 20%. A has crystal
structure available, so if I want to model protein B, what's the best
way to do it? I don't think I can just thread the sequence of B to A
structure because the number of residues are different and there  
might
be gaps and insertions in the alignment. Any suggestions of good  
programs?





--
Yu Wai Chen, PhDLecturer
King's College London, Randall Division +44-207-848-8206
New Hunt's House, Guy's Campus, London SE1 1UL, U.K.


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] homology modeling

[Peter Schmidtke]

Try Phyre (http://www.sbg.bio.ic.ac.uk/phyre/), it worked rather well for
some of my projects with very low sequence similarity.

Peter

On Tue, 5 May 2009 12:08:04 +0300, Anastassis Perrakis 
wrote:
> Modeller is indeed an excellent program (so are some others) but,  
> without being an expert, I was told some time ago and I am still under  
> the impression, that homology modeling with 20% identity (and many  
> insertions and deletions as you point out) is a dark art. Being able  
> to suggest the identical fold with some threading algorithm, is likely  
> all you will be able to do. A homology based model might be too much  
> to ask.
> 
> A.
> 
> On May 5, 2009, at 11:39, Chen, Yu Wai wrote:
> 
>> I would recommend Modeller
>> http://www.salilab.org/modeller/
>>
>> Wai
>>> Suppose I have two proteins A and B, they are structurally  
>>> homologous,
>>> however the sequence identity is only about 20%. A has crystal
>>> structure available, so if I want to model protein B, what's the best
>>> way to do it? I don't think I can just thread the sequence of B to A
>>> structure because the number of residues are different and there  
>>> might
>>> be gaps and insertions in the alignment. Any suggestions of good  
>>> programs?
>>>
>>
>>
>> -- 
>> Yu Wai Chen, PhDLecturer
>> King's College London, Randall Division +44-207-848-8206
>> New Hunt's House, Guy's Campus, London SE1 1UL, U.K.
> 
> P please don't print this e-mail unless you really need to
> Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
> Department of Biochemistry (B8)
> Netherlands Cancer Institute,
> Dept. B8, 1066 CX Amsterdam, The Netherlands
> Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

-- 

Peter Schmidtke

--
PhD Student at the Molecular Modeling and Bioinformatics Group
Dep. Physical Chemistry
Faculty of Pharmacy
University of Barcelona

Re: [ccp4bb] homology modeling

[Rotem Sertchook]

Hi,

With 20% identity the main problem is the sequence alignment. Usually  
programs that based only sequence data are not enough in such cases  
and you need to add additional terms to get the "correct" structure  
(e.g. threading). You can see list of such programs in our site  
http://bip.weizmann.ac.il/toolbox/structure/3d.htm


Try also  to work on the sequence alignment by doing multiple  
sequence alignment (and not just pair alignment) for selected group  
of sequences. You can use for that programs like TCoffee packege  
(http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi) ,  
Consensus Server (http://structure.bu.edu/cgi-bin/consensus/ 
consensus.cgi) and maybe to add structural information to the  
alignment (e.g. predicted secondary structure) like in Promals3D  
(http://prodata.swmed.edu/promals3d/promals3d.php)


after you have good alignment, the building is straight forward in  
any homology building program. Gap and insertion are usually in  
protein loops and they will be always the weak part of your building


Good Luck
Rotem



On 5 May, 2009, at 12:08, Anastassis Perrakis wrote:

Suppose I have two proteins A and B, they are structurally  
homologous,

however the sequence identity is only about 20%. A has crystal
structure available, so if I want to model protein B, what's the  
best

way to do it? I don't think I can just thread the sequence of B to A
structure because the number of residues are different and there  
might
be gaps and insertions in the alignment. Any suggestions of good  
programs?





--
Yu Wai Chen, PhDLecturer
King's College London, Randall Division +44-207-848-8206
New Hunt's House, Guy's Campus, London SE1 1UL, U.K.


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28  
597791









 
---

Rotem Sertchook, Ph.D.
Bioinformatics Unit, Biological Services
Weizmann Institute of Science,
Rehovot 76100, Israel.
 

Re: [ccp4bb] uniquefy and CELL dimension swap

[Eleanor Dodson]

I think the problem is not in uniqueify but in the GUI implementation.

to run uniqueify you need this sort of information

SYMM P212121
CELL 10 20 30 90 90 90
RESO 2.3

The GUI helpfully tries to extract the information from an existing 
reflection file and obviously can get it wrong..


You could use the option - run and view command file and edit the 
required  cell into the uniqueify part..


Eleanor




hari jayaram wrote:

I am using ccp4-6.1.1 on linux and OSX .

I have a problem with uniquefy adopting the cell dimensions of the "wrong"
data set during an rfree copy.

When I use uniquefy to copy an rfree column from one data set to another. Is
it normal for uniquefy to replace the cell dimensions in the data to the
cell dimensions from the mtz that should merely be providing the rfree
column.

Thank you for your help in advance
Hari

  

[ccp4bb] issues with idiffdisp

[James Foadi]

I have tried starting "idiffdisp"" from ccp4i, but got a wish window open and a 
message saying:
"Impossible to load Diffraction Image library 
/home/james/build/ccp4-6.1.1/lib/libDiffractionImage.so!! ..."

The file is there, though:

> ls -l $CCP4_LIB
.   .   .   .   .   .   .   .   .
.   .   .   .   .   .   .   .   .
.   .   .   .   .   .   .   .   .
-rw-r--r--  1 james james 2974782 2009-02-25 18:14 libDiffImage.a
-rwxr-xr-x  1 james james 773 2009-02-25 18:14 libDiffImage.la
-rw-r--r--  1 james james 3120264 2009-02-25 18:14 lib_DiffractionImage.a
-rw-r--r--  1 james james 3076274 2009-02-25 18:14 libDiffractionImage.a
-rwxr-xr-x  1 james james 901 2009-02-25 18:14 lib_DiffractionImage.la
-rwxr-xr-x  1 james james 894 2009-02-25 18:14 libDiffractionImage.la
lrwxrwxrwx  1 james james  29 2009-02-25 18:14 lib_DiffractionImage.so -> 
lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 james james  28 2009-02-25 18:14 libDiffractionImage.so -> 
libDiffractionImage.so.0.0.0
lrwxrwxrwx  1 james james  29 2009-02-25 18:14 lib_DiffractionImage.so.0 -> 
lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 james james  28 2009-02-25 18:14 libDiffractionImage.so.0 -> 
libDiffractionImage.so.0.0.0
-rwxr-xr-x  1 james james 4042747 2009-02-25 18:14 lib_DiffractionImage.so.0.0.0
-rwxr-xr-x  1 james james 4015515 2009-02-25 18:14 libDiffractionImage.so.0.0.0
.   .   .   .   .   .   .   .   .
.   .   .   .   .   .   .   .   .
.   .   .   .   .   .   .   .   .


I know nothing about Tcl, so I don't know how to solve this issue. Any idea on 
this from somebody else would be very much appreciated.


J

 Dr James Foadi PhD
Membrane Protein Laboratory
Diamond Light Source Ltd.
Diamond House
Harwell Science and Innovation Campus
Didcot
Oxfordshire
OX11 0DE
United Kingdom


office email: james.fo...@diamond.ac.uk
alternative email: j.fo...@imperial.ac.uk

[ccp4bb] Refolding of Denatured Protein

[Sanjiv Kumar]

I am working on a membrane protein. There was problem with the normal
purification of the protein so I have tired to purify it under denaturing
conditions (Using 8M Urea). Luckily I could purify the protein in large
quantity. But now the problem is that I am not able to refold the protein by
step wise removing 8M Urea by dialysis (8M, 6M, 4M, 2M, 1M, 0.5M and 0M
urea). It is showing aggregation at very high urea concentration say 2M
urea. Kindly suggest any alternate method that I can try for refolding of
this protein. It is a prokaryotic protein, cloned in pET28a and expressed in
BL21 DE3. Please Help.

Sanjiv Kumar

[ccp4bb] AW: [ccp4bb] Refolding of Denatured Protein

[Jan Schoepe]

Urea can modify your protein. Maybe you should also try guanidine HCl. Also, 
did you try any detergents? Good luck! Jan

--- Sanjiv Kumar  schrieb am Di, 5.5.2009:
Von: Sanjiv Kumar 
Betreff: [ccp4bb] Refolding of Denatured Protein
An: CCP4BB@JISCMAIL.AC.UK
Datum: Dienstag, 5. Mai 2009, 13:27

I am working on a membrane protein. There was problem with the normal
purification of the protein so I have tired to purify it under
denaturing conditions (Using 8M Urea). Luckily I could purify the
protein in large quantity. But now the problem is that I am not able to
refold the protein by step wise removing 8M Urea by dialysis (8M, 6M,
4M, 2M, 1M, 0.5M and 0M urea). It is showing aggregation at very high
urea concentration say 2M urea. Kindly suggest any alternate method that I can 
try for
refolding of this protein. It is a prokaryotic protein, cloned in
pET28a and expressed in BL21 DE3. Please Help.


Sanjiv Kumar 



  

Re: [ccp4bb] Refolding of Denatured Protein

[Sanjiv Kumar]

My protein mainly has alpha helices and very few beta barrel as predicted by
PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/psiform.html). There are no
literature available on this protein so I can not be very sure. I have not
tried purifying this protein either with detergent or with *Guanidine
Hydrochloride. I can try with both *Guanidine HCl and detergent. Since I got
good purification with the 8M urea, I was thinking if this protein could be
refolded and used further. Isn't removal of detergent more difficult then
Urea?

Sanjiv Kumar
Lab. No. 411,
Functional Genomics Unit,
Institute of Genomics and Integrative Biology,
New Delhi-110007
India


On Tue, May 5, 2009 at 5:27 PM, vinothkumar wrote:

> Is it a beta barrel or alpha helical protein? If it is a beta barrel then
> there is lot of information available, I hope that when you were removing
> urea you used some detergent and at some concentration higher than CMC
> (generally for refolding you should use more 3-4 times CMC) that is because
> in urea (depending on conc.) the CMC differs. For alpha helical protein it
> is bit more difficult because of their hydrophobic nature, they may require
> specific lipids for assembly. There aren't  very many examples of alpha
> helical protein (with many TM helices) refolded and structure solved.  Hope
> this helps.
>
>
> Vinoth
>
> On 5 May 2009, at 12:27, Sanjiv Kumar wrote:
>
>  I am working on a membrane protein. There was problem with the normal
>> purification of the protein so I have tired to purify it under denaturing
>> conditions (Using 8M Urea). Luckily I could purify the protein in large
>> quantity. But now the problem is that I am not able to refold the protein by
>> step wise removing 8M Urea by dialysis (8M, 6M, 4M, 2M, 1M, 0.5M and 0M
>> urea). It is showing aggregation at very high urea concentration say 2M
>> urea. Kindly suggest any alternate method that I can try for refolding of
>> this protein. It is a prokaryotic protein, cloned in pET28a and expressed in
>> BL21 DE3. Please Help.
>>
>> Sanjiv Kumar
>>
>
> Vinothkumar K.R.
> Structural Studies Division
> MRC-Laboratory of Molecular Biology
> Hills Road, Cambridge.
> CB2 0QH
>
> Tel: 44-1223-402405
>
>
>
>
>
>

Re: [ccp4bb] Refolding of Denatured Protein

[David Cobessi]

Sanjiv Kumar wrote:
I am working on a membrane protein. There was problem with the normal 
purification of the protein so I have tired to purify it under 
denaturing conditions (Using 8M Urea). Luckily I could purify the 
protein in large quantity. But now the problem is that I am not able 
to refold the protein by step wise removing 8M Urea by dialysis (8M, 
6M, 4M, 2M, 1M, 0.5M and 0M urea). It is showing aggregation at very 
high urea concentration say 2M urea. Kindly suggest any alternate 
method that I can try for refolding of this protein. It is a 
prokaryotic protein, cloned in pET28a and expressed in BL21 DE3. 
Please Help.


Sanjiv Kumar 

Dear Sanjiv,
I guess that the protein is well expressed in the membranes if there is 
only (if I could say) a problem with the purification, and that you are 
also able to solubilize the membranes and extract the protein with 
detergents.
Have you tried several detergents for the purification? Or several 
buffers with or without salt?

David

--
David Cobessi
Institut de Biologie Structurale
41, Rue Jules Horowitz
38027 Grenoble Cedex-1, France
Tel:33(0)438789613
   33(0)608164340
Fax:33(0)438785122 

Re: [ccp4bb] Refolding of Denatured Protein

[David Cobessi]

Sanjiv Kumar wrote:
 I have not tried purifying this protein either with detergent or with 
/Guanidine Hydrochloride. I can try with both /Guanidine HCl and 
detergent. Since I got good purification with the 8M urea, I was 
thinking if this protein could be refolded and used further. Isn't 
removal of detergent more difficult then Urea?

Sanjiv Kumar
Lab. No. 411,
Functional Genomics Unit,
Institute of Genomics and Integrative Biology,
New Delhi-110007
India
Is it a transmembrane protein or not? or just a protein associated to 
the membrane (with a GPI anchor for example)?
If it is a transmembrane protein, you should try to use detergents for 
extraction and during the purification.

David

--
David Cobessi
Institut de Biologie Structurale
41, Rue Jules Horowitz
38027 Grenoble Cedex-1, France
Tel:33(0)438789613
   33(0)608164340
Fax:33(0)438785122 

Re: [ccp4bb] Refolding of Denatured Protein

[Sanjiv Kumar]

This protein does not have any trans membrane helices, as predicted by TMHMM
server (http://www.cbs.dtu.dk/services/TMHMM/).  Protein is predicted to be
outside the cell by TMHMM. This protein is not there in the soluble
fraction, but it is in the cell pellet. I will try purification with the
detergents. Hope this works. Thanks a lot for help.

On Tue, May 5, 2009 at 6:08 PM, vinothkumar wrote:

> If it is really a membrane protein I am afraid you have to use detergent
> for purification. Since you did not use detergent the protein did not elute
> of the column, the first thing to try is to solubilize your protein in
> detergent (that is from membranes) and purify in presence of detergent. This
> way you don't have to worry about refolding. How many TM helices does your
> protein has?
>
>
> Vinothkumar K.R.
> Structural Studies Division
> MRC-Laboratory of Molecular Biology
> Hills Road, Cambridge.
> CB2 0QH
>
> Tel: 44-1223-402405
>
>
>
>
>
>


-- 

Sanjiv Kumar
Lab. No. 411,
Functional Genomics Unit,
Institute of Genomics and Integrative Biology,
New Delhi-110007
India

Re: [ccp4bb] Refolding of Denatured Protein

[Shao-Yang Ku]

Your protein concentration may be too high, and the stepwise dilution  
or dialysis may give your semi-folded protein plenty of chance to  
aggregate. I had better luck with rapid dilution of unfolded protein  
in large quantity of buffer in the presence of detergents ($$$).  
(Naturally you would try to use the cheapest detergent first). After  
concentrating your protein, you may recycle your refolding buffer if  
cost has become an issue. Many poly-ol compounds in high concentration  
also suppress aggregation. However, the absence of aggregates doesn't  
mean your protein is correctly folded. An activity assay is essential.  
Also be aware that some anionic detergents will precipitate with  
guanidinium chloride if you choose it as your denaturant. It also  
shifts the standard curve of your colorimetric method that you may use  
to estimate the protein concentration.


Good Luck,

SY

Quoting "Sanjiv Kumar" :


I am working on a membrane protein. There was problem with the normal
purification of the protein so I have tired to purify it under denaturing
conditions (Using 8M Urea). Luckily I could purify the protein in large
quantity. But now the problem is that I am not able to refold the protein by
step wise removing 8M Urea by dialysis (8M, 6M, 4M, 2M, 1M, 0.5M and 0M
urea). It is showing aggregation at very high urea concentration say 2M
urea. Kindly suggest any alternate method that I can try for refolding of
this protein. It is a prokaryotic protein, cloned in pET28a and expressed in
BL21 DE3. Please Help.

Sanjiv Kumar

Re: [ccp4bb] Refolding of Denatured Protein

[Sanjiv Kumar]

Yes, it could be the reason that protein concentration is high. The SDS-PAGE
shows big blob of purified protein of probable size. I can try rapid
dilution method.

Since it is in 8M Urea I have not estimated it yet by any protein estimation
assay, but I am sure that protein concentration is high.

I wish to know how to proceed after rapid dilution,  say 8M Urea to 0.8M
Urea in one step and after that? How do I go from 0.8M to 0M urea, here I
have to go gradually? or stepwise? or again rapid dilution? This reduced the
concentration of the protein. That is not very big problem as I have
concentrators.

Thanks a lot for help and suggestions.

Sanjiv Kumar
Lab. No. 411,
Functional Genomics Unit,
Institute of Genomics and Integrative Biology,
New Delhi-110007
India

On Tue, May 5, 2009 at 6:40 PM, Shao-Yang Ku  wrote:

> Your protein concentration may be too high, and the stepwise dilution or
> dialysis may give your semi-folded protein plenty of chance to aggregate. I
> had better luck with rapid dilution of unfolded protein in large quantity of
> buffer in the presence of detergents ($$$). (Naturally you would try to use
> the cheapest detergent first). After concentrating your protein, you may
> recycle your refolding buffer if cost has become an issue. Many poly-ol
> compounds in high concentration also suppress aggregation. However, the
> absence of aggregates doesn't mean your protein is correctly folded. An
> activity assay is essential. Also be aware that some anionic detergents will
> precipitate with guanidinium chloride if you choose it as your denaturant.
> It also shifts the standard curve of your colorimetric method that you may
> use to estimate the protein concentration.
>
> Good Luck,
>
> SY
>
>
> Quoting "Sanjiv Kumar" :
>
>  I am working on a membrane protein. There was problem with the normal
>> purification of the protein so I have tired to purify it under denaturing
>> conditions (Using 8M Urea). Luckily I could purify the protein in large
>> quantity. But now the problem is that I am not able to refold the protein
>> by
>> step wise removing 8M Urea by dialysis (8M, 6M, 4M, 2M, 1M, 0.5M and 0M
>> urea). It is showing aggregation at very high urea concentration say 2M
>> urea. Kindly suggest any alternate method that I can try for refolding of
>> this protein. It is a prokaryotic protein, cloned in pET28a and expressed
>> in
>> BL21 DE3. Please Help.
>>
>> Sanjiv Kumar
>>
>>


-- 

Sanjiv Kumar
Lab. No. 411,
Functional Genomics Unit,
Institute of Genomics and Integrative Biology,
New Delhi-110007
India

Re: [ccp4bb] Refolding of Denatured Protein

[Ashley Buckle]

Have a look at REFOLD: http://refold.med.monash.edu.au/

cheers
Ashley

On 05/05/2009, at 9:27 PM, Sanjiv Kumar wrote:

I am working on a membrane protein. There was problem with the  
normal purification of the protein so I have tired to purify it  
under denaturing conditions (Using 8M Urea). Luckily I could purify  
the protein in large quantity. But now the problem is that I am not  
able to refold the protein by step wise removing 8M Urea by dialysis  
(8M, 6M, 4M, 2M, 1M, 0.5M and 0M urea). It is showing aggregation at  
very high urea concentration say 2M urea. Kindly suggest any  
alternate method that I can try for refolding of this protein. It is  
a prokaryotic protein, cloned in pET28a and expressed in BL21 DE3.  
Please Help.


Sanjiv Kumar




Associate Professor Ashley M Buckle
NHMRC Senior Research Fellow
The Department of Biochemistry and Molecular Biology
School of Biomedical Sciences, Faculty of Medicine &
Victorian Bioinformatics Consortium (VBC)
Monash University, Clayton, Vic 3800
Australia

http://www.med.monash.edu.au/biochem/staff/abuckle.html
iChat/AIM: blindcaptaincat
skype: ashley.buckle
Tel: (613) 9902 9313 (office)
Fax : (613) 9905 4699

[ccp4bb] research position at Glaxo SmithKline --North Carolina

[Annie Hassell]

Preferred Qualifications 
 
This position requires a BA or BS in Chemistry, Biochemistry, or other 
related major with at least 5 years of molecular biology or biochemistry 
experience, or MS degree in a relevant area plus typically a minimum of 3 
years relevant experience. Experience with molecular biology and protein 
purification is required. Good communication skills, the ability to 
effectively interact with others in a matrix organization, and the ability 
to independently plan and direct own assignments to successful completion 
are also essential. Experience with protein biochemistry and 
crystallization is desirable. Experience in using robotics for performing 
crystallization experiments and imaging results is a strong plus.








Details 
 
Overview
The GlaxoSmithkline Structural Sciences group in Molecular Discovery 
Research at RTP, NC is seeking an associate scientist to apply molecular 
and structural biology approaches to the discovery of new drugs.

Job objective
The incumbent will work with a multidisciplinary team representing 
biochemistry, medicinal chemistry, and structural biology. An integral 
part of this position is to utilize cloning, protein expression and 
purification, and other relevant biochemical techniques, to obtain 
crystals of protein/drug complexes for structural determination. This 
position involves improvement of established systems, as well as 
developing protocols for characterizing and crystallizing new drug 
targets.

All applications must be made at the Web site

http://us.gsk.com/html/career/index.html

Job requisition #53868

Re: [ccp4bb] issues with idiffdisp

[James Foadi]

It doesn't work outside the GIU either.
I should tell that I have my CCP4 install compiled from source, it is not a 
binary installation.
Also that I use ActiveTcl 8.4 and my Linux platform is Ubuntu 8 (64 bit). I 
wonder whether
this is a Tcl version issue...

J

 Dr James Foadi PhD
Membrane Protein Laboratory
Diamond Light Source Ltd.
Diamond House
Harwell Science and Innovation Campus
Didcot
Oxfordshire
OX11 0DE
United Kingdom


office email: james.fo...@diamond.ac.uk
alternative email: j.fo...@imperial.ac.uk
web page:





From: Boaz Shaanan 
To: James Foadi 
Sent: Tuesday, 5 May, 2009 12:37:17
Subject: Re: [ccp4bb] issues with idiffdisp

Hi,

I use it routinely with no problem OUTSIDE the gui.

Cheers,

   Boaz 

- Original Message -
From: James Foadi 
Date: Tuesday, May 5, 2009 14:02
Subject: [ccp4bb] issues with idiffdisp
To: CCP4BB@JISCMAIL.AC.UK

> I have tried starting "idiffdisp"" from ccp4i, but got a wish 
> window open and a message saying:
> "Impossible to load Diffraction Image library 
> /home/james/build/ccp4-6.1.1/lib/libDiffractionImage.so!! ..."
> 
> The file is there, though:
> 
> > ls -l $CCP4_LIB
> .   .   .   .   
> .   .   .   .   .
> .   .   .   .   
> .   .   .   .   .
> .   .   .   .   
> .   .   .   .   .
> -rw-r--r--  1 james james 2974782 2009-02-25 18:14 libDiffImage.a
> -rwxr-xr-x  1 james james 773 2009-
> 02-25 18:14 libDiffImage.la
> -rw-r--r--  1 james james 3120264 2009-02-25 18:14 
> lib_DiffractionImage.a-rw-r--r--  1 james james 3076274 
> 2009-02-25 18:14 libDiffractionImage.a
> -rwxr-xr-x  1 james james 901 2009-
> 02-25 18:14 lib_DiffractionImage.la
> -rwxr-xr-x  1 james james 894 2009-
> 02-25 18:14 libDiffractionImage.la
> lrwxrwxrwx  1 james james  29 
> 2009-02-25 18:14 lib_DiffractionImage.so -> 
> lib_DiffractionImage.so.0.0.0lrwxrwxrwx  1 james 
> james  28 2009-02-25 18:14 
> libDiffractionImage.so -> libDiffractionImage.so.0.0.0
> lrwxrwxrwx  1 james james  29 
> 2009-02-25 18:14 lib_DiffractionImage.so.0 -> 
> lib_DiffractionImage.so.0.0.0lrwxrwxrwx  1 james 
> james  28 2009-02-25 18:14 
> libDiffractionImage.so.0 -> libDiffractionImage.so.0.0.0
> -rwxr-xr-x  1 james james 4042747 2009-02-25 18:14 
> lib_DiffractionImage.so.0.0.0-rwxr-xr-x  1 james james 
> 4015515 2009-02-25 18:14 libDiffractionImage.so.0.0.0
> .   .   .   .   
> .   .   .   .   .
> .   .   .   .   
> .   .   .   .   .
> .   .   .   .   
> .   .   .   .   .
> 
> 
> I know nothing about Tcl, so I don't know how to solve this 
> issue. Any idea on this from somebody else would be very much 
> appreciated.
> 
> J
> 
>  Dr James Foadi PhD
> Membrane Protein Laboratory
> Diamond Light Source Ltd.
> Diamond House
> Harwell Science and Innovation Campus
> Didcot
> Oxfordshire
> OX11 0DE
> United Kingdom
> 
> 
> office email: james.fo...@diamond.ac.uk
> alternative email: j.fo...@imperial.ac.uk
> 
> 
>   
> 

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
Phone: 972-8-647-2220 ; Fax: 646-1710
Skype: boaz.shaanan‎ 


  

[ccp4bb] Lost my protein

[yanming Zhang]

Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter devices, 
5000g, 4C, I lost large amount of my protein (75%). I heard the same story from 
one of my colleagues too. It seems the membrane of the Amicon tube ate my 
protein. 
Why this happen and how to recover my protein? 

Thank you very much.

Yan



  

Re: [ccp4bb] Lost my protein

[mesters]

Hi,

assuming that 1) you used the right MW cut-off and 2) the membrane is 
intact, you probably "pelleted" your protein onto the membrane.
This often happens if the protein has a limited solubility in the buffer 
you are using.


Remember, while concentrating your protein, you are actually "traveling" 
through the solubility phase diagram.


- J. -






yanming Zhang wrote:

Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter 
devices, 5000g, 4C, I lost large amount of my protein (75%). I heard 
the same story from one of my colleagues too. It seems the membrane of 
the Amicon tube ate my protein.

Why this happen and how to recover my protein?

Thank you very much.

Yan





--
Dr. Jeroen R. Mesters
Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
Institut für Biochemie, Universität zu Lübeck
Zentrum für Medizinische Struktur- und Zellbiologie
Ratzeburger Allee 160, D-23538 Lübeck
Tel: +49-451-5004065, Fax: +49-451-5004068
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.selfish-brain.org
Http://www.opticryst.org
--
If you can look into the seeds of time and say
which grain will grow and which will not - speak then to me  (Macbeth)
--

Re: [ccp4bb] Lost my protein

[Jacob Keller]

Check the speed--for the 15mL devices, ~3000 x g is the cutoff, I think. You 
may be breaking the membranes, and your protein is simply in the bottom of the 
concentrator.

JPK

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

  - Original Message - 
  From: yanming Zhang 
  To: CCP4BB@JISCMAIL.AC.UK 
  Sent: Tuesday, May 05, 2009 11:06 AM
  Subject: [ccp4bb] Lost my protein


Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter 
devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same 
story from one of my colleagues too. It seems the membrane of the Amicon tube 
ate my protein. 
Why this happen and how to recover my protein? 

Thank you very much.

Yan
   

Re: [ccp4bb] failed compilation for Pointless

[James Foadi]

For those of you who have been experiencing my same problem, I have solved it.
It just sufficed downloading and installing the patch (source code bit):
  
http://www.ccp4.ac.uk/updates/

The compilation went too fast for me to be able to read quickly through the 
lines, but I could see that some
of it involved fftw, I suspect the real problem in my failed previous attempt.
I can now run the full Pointless without problems.

J

 Dr James Foadi PhD
Membrane Protein Laboratory
Diamond Light Source Ltd.
Diamond House
Harwell Science and Innovation Campus
Didcot
Oxfordshire
OX11 0DE
United Kingdom


office email: james.fo...@diamond.ac.uk
alternative email: j.fo...@imperial.ac.uk

Re: [ccp4bb] Lost my protein

[Kornelius Zeth]

Hi Yanming,

you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep 
proteins in solution.

Best wishes

Kornelius

On Tue, 5 May 2009 09:06:09 -0700
 yanming Zhang  wrote:
> Hi all,
> 
> during concentrating my protein, using Amicon Ultra centrifugal filter 
> devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same 
> story from one of my colleagues too. It seems the membrane of the Amicon tube 
> ate my protein. 
> Why this happen and how to recover my protein? 
> 
> Thank you very much.
> 
> Yan
> 
> 
> 
>   

 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 kornelius.z...@tuebingen.mpg.de
 Tel -49 7071 601 323
 Fax -49 7071 601 349

[ccp4bb] Ecoli K12 SB536

[aka akaka]

Hello All:
 I would like to know if anyone has or knows how to gte this Ecoli strain which 
proved to be good for periplasmic protein expression. Ecoli K12 SB 536.
If not, which other strain is good for that?
Thannks Much
Dr. R. Depetris
Weill Cornell Medical College
_
See how Windows® connects the people, information, and fun that are part of 
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Re: [ccp4bb] Lost my protein

[Joe]

Hi Yanming,

I usually keep the follow through before I know nothing is in it. Also
you may want to suspend your protein solution every 5-10 min to avoid
precipitation because the concentration gradient forms rapidly during
centrifugation. If all of these do not help, just try concentrators
with different types of membrane.

Joe



Kornelius Zeth wrote:

  Hi Yanming,

you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep proteins in solution.

Best wishes

Kornelius

On Tue, 5 May 2009 09:06:09 -0700
 yanming Zhang  wrote:
  
  
Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same story from one of my colleagues too. It seems the membrane of the Amicon tube ate my protein. 
Why this happen and how to recover my protein? 

Thank you very much.

Yan



  

  
  
 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 kornelius.z...@tuebingen.mpg.de
 Tel -49 7071 601 323
 Fax -49 7071 601 349
  

[ccp4bb] desktop models

[Christopher Rife]

Hi,

I am looking to have a model produced from a PDB, i.e. something that we 
can put on the desk for everyone to admire :)

Googling for such a thing is rather challenging, so I'm curious if anyone 
has suggestions as to where I might get something like that made. So far, 
I have found two options:
1. Molecular Dimensions: 
http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2
(etching in a glass block)

2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
(nylon models)

I think #2 is more what we have in mind. Does anyone have any experience 
with their final product in terms of quality and durability?

Thanks very much.

Chris


___

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are not the intended recipient and you
receive this e-mail by mistake, you are not allowed to use the information, to 
copy it or distribute it further.
Please notify us and return it to Danisco by e-mail and delete all attachments. 
Thank you for your assistance.

__

Re: [ccp4bb] desktop models

[Schubert, Carsten [PRDUS]]

Chris,

For the laser-etched glass blocks have a look at:

http://www.bathsheba.com/

I have a couple of them and they look very good.

Cheers,

Carsten


> -Original Message-
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
> Christopher Rife
> Sent: Tuesday, May 05, 2009 2:42 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] desktop models
> 
> Hi,
> 
> I am looking to have a model produced from a PDB, i.e. something that
> we
> can put on the desk for everyone to admire :)
> 
> Googling for such a thing is rather challenging, so I'm curious if
> anyone
> has suggestions as to where I might get something like that made. So
> far,
> I have found two options:
> 1. Molecular Dimensions:
> http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2
> (etching in a glass block)
> 
> 2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
> (nylon models)
> 
> I think #2 is more what we have in mind. Does anyone have any
> experience
> with their final product in terms of quality and durability?
> 
> Thanks very much.
> 
> Chris
> 
>
___
> _
> ___
> 
> This is an e-mail from Danisco and may contain confidential
> information. If you
> are not the intended recipient and you
> receive this e-mail by mistake, you are not allowed to use the
> information, to
> copy it or distribute it further.
> Please notify us and return it to Danisco by e-mail and delete all
> attachments.
> Thank you for your assistance.
>
___
> _
> __

Re: [ccp4bb] desktop models

[Prof. Joel L. Sussman]

5-May-2009   22:00 Rehovot
Dear Chris,
   See the the page "A Physical Model of the β2-Adrenergic Receptor"  
in Proteopedia at:
http://www.proteopedia.org/wiki/index.php/A_Physical_Model_of_the_β2-Adrenergic_Recepto 
r
   You can get these kind of models from the MSOE Center for  
BioMolecular Modeling

at: http://www.rpc.msoe.edu/cbm/
best regards
Joel
-
Prof. Joel L. Sussmanjoel.suss...@weizmann.ac.il
Pickman Prof. of Structural Biology  +972 (8) 934 4531 - tel
Department of Structural Biology +972 (8) 934 4159 - fax
Weizmann Institute of Sciencewww.weizmann.ac.il/~joel
Rehovot 76100 ISRAEL www.weizmann.ac.il/ISPC

Proteopedia, www.proteopedia.org  (because life has more than 2D)
-

On 5 May 2009, at 21:41, Christopher Rife wrote:


Hi,

I am looking to have a model produced from a PDB, i.e. something  
that we

can put on the desk for everyone to admire :)

Googling for such a thing is rather challenging, so I'm curious if  
anyone
has suggestions as to where I might get something like that made. So  
far,

I have found two options:
1. Molecular Dimensions:
http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2
(etching in a glass block)

2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
(nylon models)

I think #2 is more what we have in mind. Does anyone have any  
experience

with their final product in terms of quality and durability?

Thanks very much.

Chris


___

This is an e-mail from Danisco and may contain confidential  
information. If you

are not the intended recipient and you
receive this e-mail by mistake, you are not allowed to use the  
information, to

copy it or distribute it further.
Please notify us and return it to Danisco by e-mail and delete all  
attachments.

Thank you for your assistance.

__

Re: [ccp4bb] desktop models

[Raji Edayathumangalam]

I've seen some very pretty 3D models in optical crystal glass made  
and sold by

http://www.luminorum.com/

Cheers,
Raji
Disclaimer: I have nothing to do with this company.



On May 5, 2009, at 2:41 PM, Christopher Rife wrote:


Hi,

I am looking to have a model produced from a PDB, i.e. something  
that we

can put on the desk for everyone to admire :)

Googling for such a thing is rather challenging, so I'm curious if  
anyone
has suggestions as to where I might get something like that made.  
So far,

I have found two options:
1. Molecular Dimensions:
http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2
(etching in a glass block)

2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
(nylon models)

I think #2 is more what we have in mind. Does anyone have any  
experience

with their final product in terms of quality and durability?

Thanks very much.

Chris

__ 
__

___

This is an e-mail from Danisco and may contain confidential  
information. If you

are not the intended recipient and you
receive this e-mail by mistake, you are not allowed to use the  
information, to

copy it or distribute it further.
Please notify us and return it to Danisco by e-mail and delete all  
attachments.

Thank you for your assistance.
__ 
__

__

Re: [ccp4bb] Lost my protein

[Matthew Alan Bratkowski]

Hi.

I have experienced similar problems with the Amicon filters previously.  I
found that using a swing bucket rather than a fixed angle centrifuge and
keeping the speed at 4000 g, the recommended speed, resulted in better
yields.  If you have a large volume to concentrate (>12 mL), using an
Amicon stirred pressure cell first may work better.  This prevents
repeated spinning of the filter, which is not recommended by the company. 
In my experience, using the same filter a few times is ok, but I guess
that repeated centrifugation could eventually crack the filter.

Matt

Re: [ccp4bb] desktop models

[Christopher Bahl]

There's also:
http://bioetch.com/

-Chris


Raji Edayathumangalam wrote:
I've seen some very pretty 3D models in optical crystal glass made and 
sold by

http://www.luminorum.com/

Cheers,
Raji
Disclaimer: I have nothing to do with this company.



On May 5, 2009, at 2:41 PM, Christopher Rife wrote:


Hi,

I am looking to have a model produced from a PDB, i.e. something that we 
can put on the desk for everyone to admire :)


Googling for such a thing is rather challenging, so I'm curious if 
anyone 
has suggestions as to where I might get something like that made. So 
far, 
I have found two options:
1. Molecular Dimensions: 
http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2 


(etching in a glass block)

2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
(nylon models)

I think #2 is more what we have in mind. Does anyone have any experience 
with their final product in terms of quality and durability?


Thanks very much.

Chris


___

This is an e-mail from Danisco and may contain confidential 
information. If you 
are not the intended recipient and you
receive this e-mail by mistake, you are not allowed to use the 
information, to 
copy it or distribute it further.
Please notify us and return it to Danisco by e-mail and delete all 
attachments. 
Thank you for your assistance.


__

Re: [ccp4bb] desktop models

[Andreas Förster]

Just buy this cheapo little printer and make your own.
http://www.rap-man.com/

Andreas



Christopher Rife wrote:

Hi,

I am looking to have a model produced from a PDB, i.e. something that we 
can put on the desk for everyone to admire :)


Googling for such a thing is rather challenging, so I'm curious if anyone 
has suggestions as to where I might get something like that made. So far, 
I have found two options:
1. Molecular Dimensions: 
http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2

(etching in a glass block)

2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
(nylon models)

I think #2 is more what we have in mind. Does anyone have any experience 
with their final product in terms of quality and durability?


Thanks very much.

Chris


___

This is an e-mail from Danisco and may contain confidential information. If you 
are not the intended recipient and you
receive this e-mail by mistake, you are not allowed to use the information, to 
copy it or distribute it further.
Please notify us and return it to Danisco by e-mail and delete all attachments. 
Thank you for your assistance.


__



--
Andreas Förster, Research Associate
Paul Freemont & Xiaodong Zhang Labs
Department of Biochemistry, Imperial College London
http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] desktop models

[mark michaels]

And another DIY version,
http://homemade3dprinter.blogspot.com/

m

> From: docandr...@gmail.com
> Subject: Re: [ccp4bb] desktop models
> 
> Just buy this cheapo little printer and make your own.
> http://www.rap-man.com/
> 
> Andreas
> 
> Christopher Rife wrote:
> > Hi,
> > 
> > I am looking to have a model produced from a PDB, i.e. something that we 
> > can put on the desk for everyone to admire :)



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Re: [ccp4bb] Lost my protein

[Jan Jensen]

When using spin concentrators, always remember to not spin more than 4-6min
before remixing the content of the concentrator by pippetting, to prevent
concentration gradient and subsequent precipitation at the bottom.
Jan


Jan K Jensen, Pl-R, Ph.D. 
Post doctoral fellow at University of Illinois at Chicago (UIC)
Laboratory of Peter GW Gettins
Department of Biochemistry and Molecular Genetics
MBRB Room 1260
900 S Ashland
Chicago Il, 60607


-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Matthew Alan Bratkowski
Sent: Tuesday, May 05, 2009 2:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Lost my protein


Hi.

I have experienced similar problems with the Amicon filters previously.  I
found that using a swing bucket rather than a fixed angle centrifuge and
keeping the speed at 4000 g, the recommended speed, resulted in better
yields.  If you have a large volume to concentrate (>12 mL), using an Amicon
stirred pressure cell first may work better.  This prevents repeated
spinning of the filter, which is not recommended by the company. 
In my experience, using the same filter a few times is ok, but I guess that
repeated centrifugation could eventually crack the filter.

Matt

Re: [ccp4bb] homology modeling

[Donnie Berkholz]

On 12:08 Tue 05 May , Anastassis Perrakis wrote:
> Modeller is indeed an excellent program (so are some others) but,  
> without being an expert, I was told some time ago and I am still under  
> the impression, that homology modeling with 20% identity (and many  
> insertions and deletions as you point out) is a dark art. Being able to 
> suggest the identical fold with some threading algorithm, is likely all 
> you will be able to do. A homology based model might be too much to ask.

Try HHpred. It will do a profile-profile alignment (well, technically 
HMM-HMM) and optionally create a multiple-template-based model using 
Modeller, all with very little knowledge or input required on the user 
end. Evaluating the likely correctness afterwards is a different 
story...

-- 
Thanks,
Donnie

Donnie Berkholz
P. Andrew Karplus lab
Oregon State University


pgpWMs80Q7nEF.pgp
Description: PGP signature

Re: [ccp4bb] Lost my protein

[Sophia Tsai]

Hi everyone,
It seems that our lab has experienced issues with precipitation of some
proteins (not all) with the Amicon concentrators. For those, we have started
using VivaSpin concentrators. That seems to work fairly well with those that
have issues with the filters. You may want to give it a try.

Best,
Sophia

Re: [ccp4bb] Lost my protein

[Li Sheng]

Dear Zhang,

 

 

Perhaps you can try to concentrate your protein using PEG2.

Put your protein into dialysis tubing and bury it with PEG2. You should
check it every 30-60 min.

 

 

Sincerely,

Li

 

 

 


Hi all,

during concentrating my protein, using Amicon Ultra centrifugal filter
devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the
same story from one of my colleagues too. It seems the membrane of the
Amicon tube ate my protein. 
Why this happen and how to recover my protein? 

Thank you very much.

Yan

 

Re: [ccp4bb] Lost my protein

[atul kumar]

sophia
i dont have any idea abt vivaspin concentrators,can u send some details abt 
it,sice we are also facing the precipitation problem during the concn.

thanks
atul

-Original Message-
From: CCP4 bulletin board on behalf of Sophia Tsai
Sent: Wed 5/6/2009 7:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Lost my protein
 
Hi everyone,
It seems that our lab has experienced issues with precipitation of some
proteins (not all) with the Amicon concentrators. For those, we have started
using VivaSpin concentrators. That seems to work fairly well with those that
have issues with the filters. You may want to give it a try.

Best,
Sophia

Re: [ccp4bb] problem in transformation of pqe 30 clone

[atul kumar]

now i have transformed pqe 30 clone into the m15 host successfully, can someone 
let me know exactly what was reason behind the problem into transformation??is 
it leaky expression into the other host that is toxic for the cells,if it so 
then will i would be  able to get good expression into this host???

atul
-Original Message-
From: CCP4 bulletin board on behalf of ar...@xtals.org
Sent: Tue 5/5/2009 6:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone
 
To clarify: I am not implying that I've worked with many proteins that
express better in XL1-blue cells than they would express in BL21(DE3) or
such if cloned into a pET vector or similar. In fact I can probably recall
only one or two that expressed *better* in XL1 cells. However in the good
old days pQE series of vectors was quite commonly used and I had things in
that were inherited from others - these 'things' were fairly simple to
express in XL1-blue whereas they gave me loads of trouble in other strains
and I was too busy/lazy to re-clone them.

Artem

> Hello Artem,
>
>  We express almost all our proteins in BL21 derivatives. It sounds
> like you've worked with many proteins that express/behave better in
> XL1-Blue?
>
>
> ho
> UC Berkeley
>
> -
> XL1-Blue is a strain of E. coli. Whether it is or isn't an expression host
> depends on the definition, and I am not going to argue semantics.
>
> The T5 promoter works with regular garden variety RNApol of E. coli.
> Therefore ANY E. coli strain is an 'expression host' for vectors that
> contain this promoter.
>
> I've expressed many proteins in XL1-Blue and I see no reason why you can't
> express yours, either.
>
> Artem
>

Re: [ccp4bb] Refolding of Denatured Protein

[atul kumar]

dear sanjiv
i dont think that the removal of detergent is more difficult than urea,since u 
are purifying ur protein over Ni-NTA column,so after a few wash over there u 
can remove the detergent completely.

best
atul kumar

-Original Message-
From: CCP4 bulletin board on behalf of David Cobessi
Sent: Tue 5/5/2009 6:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refolding of Denatured Protein
 
Sanjiv Kumar wrote:
>  I have not tried purifying this protein either with detergent or with 
> /Guanidine Hydrochloride. I can try with both /Guanidine HCl and 
> detergent. Since I got good purification with the 8M urea, I was 
> thinking if this protein could be refolded and used further. Isn't 
> removal of detergent more difficult then Urea?
> Sanjiv Kumar
> Lab. No. 411,
> Functional Genomics Unit,
> Institute of Genomics and Integrative Biology,
> New Delhi-110007
> India
Is it a transmembrane protein or not? or just a protein associated to 
the membrane (with a GPI anchor for example)?
If it is a transmembrane protein, you should try to use detergents for 
extraction and during the purification.
David

-- 
David Cobessi
Institut de Biologie Structurale
41, Rue Jules Horowitz
38027 Grenoble Cedex-1, France
Tel:33(0)438789613
33(0)608164340
Fax:33(0)438785122 

Re: [ccp4bb] Lost my protein

[Jobichen Chacko]

I also some times seen that proteins are not sticking that much with
vivaspin concentrrtors. These are transparent concentrators and it is easy
to collect the  concentrated protein by giving  an inverted spin for 1
minute. Please check the website for more details www.vivascience.com
Jobi


On Wed, May 6, 2009 at 3:18 PM, atul kumar  wrote:

>  sophia
> i dont have any idea abt vivaspin concentrators,can u send some details abt
> it,sice we are also facing the precipitation problem during the concn.
>
> thanks
> atul
>
> -Original Message-
> From: CCP4 bulletin board on behalf of Sophia Tsai
> Sent: Wed 5/6/2009 7:12 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Lost my protein
>
> Hi everyone,
> It seems that our lab has experienced issues with precipitation of some
> proteins (not all) with the Amicon concentrators. For those, we have
> started
> using VivaSpin concentrators. That seems to work fairly well with those
> that
> have issues with the filters. You may want to give it a try.
>
> Best,
> Sophia
>
>