[ccp4bb]

[nisha pandey]

Dear ccp4bb,
I want to know regarding Wilson B value and mean B-value of protein after
refinement, In my case there is large difference in Wilson plot and
overall.Is it OK to have such value ie Wilson B-value 42 and mean B value is
23.3 ?
Also in the same structure I have observed that in a few residues B-value of
main chain are higher than that of side chain.It seems quit unusual .Kindly
help me sort out this problem.
Some basic information regarding the data  are given below.

Resolution range 41.24-2.7
Rsym  -15
I/Sigma-17.2
I/Sigma  -4.2
(In highest resolution shell)
Rfactor -21.4
R free  -24.2

Thanks in advance

With regards
Nisha

Re: [ccp4bb] Using chaperones to boost expression in E. coli

[Shaun Lott]

As with all these things, YMMV.

In our experience, chaperone co-expression can help not at all, can produce 
more folded protein or can produce more soluble protein, with co-purifying 
chaperone. With GroEL/S at least, chaperone release is easy (add ATP) and 
sometimes results in the liberation of folded protein, sometimes not.

One case where for us chaperones made an enabling difference: Acta Cryst. 
(2005). F61, 403–406 

Like most things, worth trying!

cheers

Shaun

Re: [ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Bi-Cheng Wang]

Dear Colleagues,

Based on the note below from Gerard Bricogne, I would like to further clarify 
and highlight the intended purpose of the workshop for those of you who may be 
interested in attending. 

The planned discussions of the workshop will focus on the positive and useful 
benefits of using soft x-rays in macromolecular crystallography with 
wavelengths between 1.5 Å and 4.0 Å.  The talks will also provide participants 
with an updated picture of the current developments around the globe, and 
include several key international speakers that are currently working in this 
fast growing field of study. 

I hope to see many of you at the APS, or possibly at the ACA meeting later in 
May or IUCr in August.

Best regards,

B.C.

On Apr 22, 2011, at 12:08 PM, Gerard Bricogne wrote:

> Dear BC and colleagues,
> 
> It is rather unclear from the title of the workshop whether it is the
> Crystallography that will be soft, or the X-rays.
> 
> 
> With best wishes,
> 
>  Gerard.
> 
> --
> On Fri, Apr 22, 2011 at 11:56:22AM -0400, Bi-Cheng Wang wrote:
>> We would like to call your attention to a workshop associated with the 
>> upcoming 2011 APS Users Meeting, which will be held on May 4, 2011 at the 
>> APS, Argonne National Laboratory, USA. 
>> 
>> The title of the workshop is  “Soft X-ray Crystallography”.  You may find 
>> the workshop program at 
>> 
>> http://2011usersmeeting.conference.anl.gov/APSWK1Agenda.htm
>> 
>> The web links for meeting registration and the application for a permit to 
>> enter the Argonne National Laboratory are:
>> 
>> https://2011usersmeeting.conference.anl.gov/register
>> 
>> http://www.aps.anl.gov/Users/Site_Access
>> 
>> We hope you will be able to attend the APS Users Meeting and our workshop.
>> 
>> All the best,
>> 
>> B.C. Wang, John Rose, Gerd Rosenbaum and John Chrzas.
> -- 
> 
> ===
> * *
> * Gerard Bricogne g...@globalphasing.com  *
> * *
> * Global Phasing Ltd. *
> * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
> * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
> * *
> ===

Re: [ccp4bb] Sketcher

[Subhangi Ghosh]

You can change the target torsion angle for the double bond in the .cif file
that is created when your molecule is created.

--Subhangi


On Fri, Apr 22, 2011 at 3:11 PM, Jim Fairman  wrote:

> I am attempting to create a monomer with a cis-double bond using Sketcher,
> but every time I tell sketcher to create the library files it makes the
> double bond a trans-double bond.  Can anyone assist me with information on
> how to force it to remain cis?
>
> --
> Jim Fairman, Ph D.
> Post-Doctoral Fellow
> National Institutes of Health - NIDDK
> The Buchanan Lab 
> Lab: 1-301-594-9229
> E-mail: fairman@gmail.com james.fair...@nih.gov
>
>

[ccp4bb] Sketcher

[Jim Fairman]

I am attempting to create a monomer with a cis-double bond using Sketcher,
but every time I tell sketcher to create the library files it makes the
double bond a trans-double bond.  Can anyone assist me with information on
how to force it to remain cis?

-- 
Jim Fairman, Ph D.
Post-Doctoral Fellow
National Institutes of Health - NIDDK
The Buchanan Lab 
Lab: 1-301-594-9229
E-mail: fairman@gmail.com james.fair...@nih.gov

Re: [ccp4bb] Using chaperones to boost expression in E. coli

[aaleshin]

Hi Michael,
It worked for me with one viral enzyme but did not work with 2 others. In the 
successful case, I used Takara's chaperone plasmids to screen for the best 
composition of chaperones. The GroEL-GroES improved the yield of the soluble 
enzyme, but I got same activity (per 1L of media) as  before. The plasmid with  
chaperone tig gave the biggest increase in activity (~4-8 fold). Still, the 
yield was rather low, in a range of 1 mg of purified protein per 1L of media. I 
also had to optimize the expression time and temperature  to get best results. 
They were ~14 h at 17-20C. Interestingly, the protein failed to express in 
insect cells (was too poisonous for them), so the combination of a solubilizing 
domain, chaperones and Ecoli expression were the only available option for me.

Regards
Alex

On Apr 22, 2011, at 8:28 AM, Kothe, Michael wrote:

> Dear ccp4bb,
>  
> I am curious to hear of examples where the expression of well-behaved protein 
> was achieved by the coexpression of chaperones in E. coli. I know the 
> appropriate strains and vectors exist, but I can’t remember hearing of a 
> successful case. I have heard anecdotally of several cases where it was tried 
> without success (including one attempt I made myself). I also heard of 
> concerns that the chaperones might copurify with the (now soluble) protein of 
> interest and are difficult to remove.
>  
> Thanks,
> Michael
>  

Re: [ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Adrian Goldman]

First line up 10^9 poohbears...

Sent from my iPhone

On 22 Apr 2011, at 19:58, Jacob Keller  wrote:

> I think soft crystallography uses fuzzy logic--it's a comforting
> alternative to the usual SAD and MAD.
> 

Re: [ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Jacob Keller]

I think soft crystallography uses fuzzy logic--it's a comforting
alternative to the usual SAD and MAD.

Re: [ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Jacob Keller]

I think soft crystallography uses fuzzy logic.
JPK

On Fri, Apr 22, 2011 at 11:08 AM, Gerard Bricogne
 wrote:
> Dear BC and colleagues,
>
>     It is rather unclear from the title of the workshop whether it is the
> Crystallography that will be soft, or the X-rays.
>
>
>     With best wishes,
>
>          Gerard.
>
> --
> On Fri, Apr 22, 2011 at 11:56:22AM -0400, Bi-Cheng Wang wrote:
>> We would like to call your attention to a workshop associated with the 
>> upcoming 2011 APS Users Meeting, which will be held on May 4, 2011 at the 
>> APS, Argonne National Laboratory, USA.
>>
>> The title of the workshop is  “Soft X-ray Crystallography”.  You may find 
>> the workshop program at
>>
>> http://2011usersmeeting.conference.anl.gov/APSWK1Agenda.htm
>>
>> The web links for meeting registration and the application for a permit to 
>> enter the Argonne National Laboratory are:
>>
>> https://2011usersmeeting.conference.anl.gov/register
>>
>> http://www.aps.anl.gov/Users/Site_Access
>>
>> We hope you will be able to attend the APS Users Meeting and our workshop.
>>
>> All the best,
>>
>> B.C. Wang, John Rose, Gerd Rosenbaum and John Chrzas.
> --
>
>     ===
>     *                                                             *
>     * Gerard Bricogne                     g...@globalphasing.com  *
>     *                                                             *
>     * Global Phasing Ltd.                                         *
>     * Sheraton House, Castle Park         Tel: +44-(0)1223-353033 *
>     * Cambridge CB3 0AX, UK               Fax: +44-(0)1223-366889 *
>     *                                                             *
>     ===
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Gerard Bricogne]

Dear BC and colleagues,

 It is rather unclear from the title of the workshop whether it is the
Crystallography that will be soft, or the X-rays.


 With best wishes,
 
  Gerard.

--
On Fri, Apr 22, 2011 at 11:56:22AM -0400, Bi-Cheng Wang wrote:
> We would like to call your attention to a workshop associated with the 
> upcoming 2011 APS Users Meeting, which will be held on May 4, 2011 at the 
> APS, Argonne National Laboratory, USA. 
>  
> The title of the workshop is  “Soft X-ray Crystallography”.  You may find the 
> workshop program at 
> 
> http://2011usersmeeting.conference.anl.gov/APSWK1Agenda.htm
>  
> The web links for meeting registration and the application for a permit to 
> enter the Argonne National Laboratory are:
>  
> https://2011usersmeeting.conference.anl.gov/register
> 
> http://www.aps.anl.gov/Users/Site_Access
>  
> We hope you will be able to attend the APS Users Meeting and our workshop.
>  
> All the best,
>  
> B.C. Wang, John Rose, Gerd Rosenbaum and John Chrzas.
-- 

 ===
 * *
 * Gerard Bricogne g...@globalphasing.com  *
 * *
 * Global Phasing Ltd. *
 * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
 * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
 * *
 ===

Re: [ccp4bb] Using chaperones to boost expression in E. coli

[Yoder, Marilyn]

My collaborator was successful in purifying soluble rat phosphatidylinositol 
transfer protein in E. coli when overexpressing GroEL/ES.  They estimated ~50% 
of the protein became soluble.  We were able to solve the structure from 
protein purified using this expression construct.   We did not experience 
copurification problems.

Yoder MD, Thomas LM, Tremblay JM, Oliver RL, Yarbrough LR, Helmkamp GM Jr. 
(2001) J. Biol. Chem. 276:9246-9252.

We have tried it with other proteins, but not as successfully.

Marilyn

Marilyn D. Yoder
Division of Cell Biology & Biophysics
School of Biological Sciences
University of Missouri-Kansas City
5007 Rockhill Rd.
Kansas City, MO 64110
Phone: 816-235-1986

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kothe, 
Michael
Sent: Friday, April 22, 2011 10:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Using chaperones to boost expression in E. coli

Dear ccp4bb,

I am curious to hear of examples where the expression of well-behaved protein 
was achieved by the coexpression of chaperones in E. coli. I know the 
appropriate strains and vectors exist, but I can't remember hearing of a 
successful case. I have heard anecdotally of several cases where it was tried 
without success (including one attempt I made myself). I also heard of concerns 
that the chaperones might copurify with the (now soluble) protein of interest 
and are difficult to remove.

Thanks,
Michael

[ccp4bb] Workshop on "Soft X-ray Crystallography" at the APS, May 4, 2011

[Bi-Cheng Wang]

We would like to call your attention to a workshop associated with the upcoming 
2011 APS Users Meeting, which will be held on May 4, 2011 at the APS, Argonne 
National Laboratory, USA. 
 
The title of the workshop is  “Soft X-ray Crystallography”.  You may find the 
workshop program at 

http://2011usersmeeting.conference.anl.gov/APSWK1Agenda.htm
 
The web links for meeting registration and the application for a permit to 
enter the Argonne National Laboratory are:
 
https://2011usersmeeting.conference.anl.gov/register

http://www.aps.anl.gov/Users/Site_Access
 
We hope you will be able to attend the APS Users Meeting and our workshop.
 
All the best,
 
B.C. Wang, John Rose, Gerd Rosenbaum and John Chrzas.

[ccp4bb] Using chaperones to boost expression in E. coli

[Kothe, Michael]

Dear ccp4bb,

 

I am curious to hear of examples where the expression of well-behaved
protein was achieved by the coexpression of chaperones in E. coli. I
know the appropriate strains and vectors exist, but I can't remember
hearing of a successful case. I have heard anecdotally of several cases
where it was tried without success (including one attempt I made
myself). I also heard of concerns that the chaperones might copurify
with the (now soluble) protein of interest and are difficult to remove.

 

Thanks,

Michael

 

Re: [ccp4bb] Is there any ccp4 tool which can add a new reflection point (hkl) to the existing mtz file?

[Phil Jeffrey]

The CCP4 program UNIQUE does this.
http://www.ccp4.ac.uk/html/unique.html

"UNIQUE creates a unique list of reflections for a given unit cell with 
a given symmetry up to a specified high resolution limit. The output 
file can be used to complete a dataset (i.e. to give an MTZ file with 
all allowed reflections present whether or not data have been measured 
for them), and to give completeness information on the measured dataset."




On 4/22/11 1:22 AM, Hailiang Zhang wrote:

Hi,

Is there any ccp4 tool which can add a new reflection point (new hkl which
was not in the existing mtz file) to the present mtz file? Thanks!

Best Regards, Hailiang