Re: [ccp4bb] recommendation for heavy atoms used in lithium sulfate and ammonium sulfate

[Ronnie Berntsson]

Hi Tiancen,

I can recommend using a Halide soak (bromide or iodine). You could  
try to "exchange" part your salt with sodium bromide. Always use a  
bad looking crystal for the first soak though to see that it doesn't  
get damaged by the soak.


See for further reference:
Novel approach to phasing proteins: derivatization by short cryo- 
soaking with halides

Z. Dauter, M. Dauter and K. R. Rajashankar
Acta Cryst. (2000). D56, 232-237

Cheers,
Ronnie Berntsson

On Apr 20, 2007, at 6:26 AM, Tiancen Hu wrote:


Dear all,

Could any one recommend some heavy atoms used for crystals grown in  
0.1M tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M  
ammonium sulfate? I read from Hampton user guide of heavy atom kit  
that "high salt concentrations are not the ideal medium for heavy  
atom reactions with macromolecules", so is there any type of heavy  
atom we should avoid using? We do not have any experience in  
preparing heavy atom derivative, so any suggestions, experience or  
references will be greatly appreciated.


Thanks in advance!

Tiancen Hu
Shanghai Institute of Materia Medica


Ronnie Berntsson

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

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

[ccp4bb] further inconsistencies (was: tcltkblt and python distributions)

[o . h . weiergraeber]

Dear ccp4 developers,

when trying to do a conventional install of ccp4 (i.e. avoiding the install.sh 
script), I ran across additional problems/inconsistencies with the package:

1. In the tarball I freshly downloaded this morning, the cctbx tree under 
ccp4-6.0.2/lib was completely missing!!!

2. The BINARY.setup script checks for src/phaser, which does not exist (instead 
there are phaser-ucs and phaser-utf directories). Simply creating the phaser 
directory may resolve this, but of course the phaser installation is corrupted 
due to the missing cctbx files.

It would be nice if the responsible persons could comment on this. Is the 
conventional install no longer supported or not even checked?

Greetings
Oliver




---
 Dr. Oliver H. Weiergraeber
 Institute of Neurosciences and Biophysics
 Molecular Biophysics
 Research Centre Juelich
 D-52425 Juelich
 Germany
 Phone: +49-2461-612028
 Fax: +49-2461-612020
---

Re: [ccp4bb] further inconsistencies (was: tcltkblt and python distributions)

[Tim Grune]

Hello Oliver,

if by 'conventional' you mean
- download the source code
- ./configure OS && make && make install

I did not share your experience a couple of days ago when I did that. I used 
both the ftp and the download interface and nothing was missing.
However from from your mentioning of 'BINARY.setup' you may have meant 
installation of the binary version. I never payed attention to that file, 
though, even though I also installed a binary set on a PC running CentOS 
4.4.
All I did was change a few variable in $CCP4/include/ccp4.setup-bash:
CCP4_MASTER to the correct root of the installation
CCP4I_TCLTK   to /usr/bin to use the system wish
and remove the MANPATH entries since with an unset MANPATH bash (or man 
itself, not sure) figures out the correct path from PATH - which makes 
maintenance a lot easier.

For CentOS and Debian I also have to change bltwish to wish in the 
tcl-scripts under $CCP4/ccp4i/bin since in both systems blt comes as a 
library.

Finally I used good old g77 instead of gfortran since the latter made mosflm 
crash with a segmentation fault as soon as it tried to display an image.

Maybe you can just try to re-download it? Unless this was all for a 
different platform in which case my advise may not be applicable - sorry.

Cheers, Tim


-Original Message-

From: [EMAIL PROTECTED]

To: CCP4BB@JISCMAIL.AC.UK

Date: Fri, 20 Apr 2007 10:44:37 +0200

Subject: [ccp4bb] further inconsistencies (was: tcltkblt and python 
distributions)




Dear ccp4 developers,



when trying to do a conventional install of ccp4 (i.e. avoiding the 
install.sh script), I ran across additional problems/inconsistencies with 
the package:



1. In the tarball I freshly downloaded this morning, the cctbx tree under 
ccp4-6.0.2/lib was completely missing!!!



2. The BINARY.setup script checks for src/phaser, which does not exist 
(instead there are phaser-ucs and phaser-utf directories). Simply creating 
the phaser directory may resolve this, but of course the phaser installation 
is corrupted due to the missing cctbx files.



It would be nice if the responsible persons could comment on this. Is the 
conventional install no longer supported or not even checked?



Greetings

Oliver









---

 Dr. Oliver H. Weiergraeber

 Institute of Neurosciences and Biophysics

 Molecular Biophysics

 Research Centre Juelich

 D-52425 Juelich

 Germany

 Phone: +49-2461-612028

 Fax: +49-2461-612020

---

Re: [ccp4bb] further inconsistencies (was: tcltkblt and python distributions)

[Remacle, F (Francois)]

Hi,

Actually, cctbx on linux has two different built, because different
linuxes use different python unicode convention (ucs and utf) therefore,
since ccbtx and phaser got some python modules built. They originally
are in $CCP4/lib-ucs and $CCP4/lib-utf, but they are not inside
$CCP4/lib. The same applies for $CCP4/src/phaser which is also split
with ucs, utf tags.
If you used install.sh, the script would have performed some check on
your machine to detect which unicode convention your python is using and
would then automatically moved the appropriate phaser / cctbx at the
proper location.
If you do not use install.sh then you have to do the check by yourself.

Hope this can help

Francois Remacle
CCP4

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: 20 April 2007 09:45
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] further inconsistencies (was: tcltkblt and python
distributions)

Dear ccp4 developers,

when trying to do a conventional install of ccp4 (i.e. avoiding the
install.sh script), I ran across additional problems/inconsistencies
with the package:

1. In the tarball I freshly downloaded this morning, the cctbx tree
under ccp4-6.0.2/lib was completely missing!!!

2. The BINARY.setup script checks for src/phaser, which does not exist
(instead there are phaser-ucs and phaser-utf directories). Simply
creating the phaser directory may resolve this, but of course the phaser
installation is corrupted due to the missing cctbx files.

It would be nice if the responsible persons could comment on this. Is
the conventional install no longer supported or not even checked?

Greetings
Oliver




---
 Dr. Oliver H. Weiergraeber
 Institute of Neurosciences and Biophysics  Molecular Biophysics
Research Centre Juelich
 D-52425 Juelich
 Germany
 Phone: +49-2461-612028
 Fax: +49-2461-612020
---

Re: [ccp4bb] further inconsistencies (was: tcltkblt and python distributions)

[o . h . weiergraeber]

OK, thanks for your comments. The most important statement was that 
BINARY.install just does not work any more without manual intervention on Linux 
systems!
I recommend placing this information in an obvious location!

*BUT*
Again trying with a fresh download (this time without python since I want to 
use the system version) I am getting an error message indicating the directory 
ccp4-6.0.1/phaserbin-ucs is missing! The phaser executable is present in 
ccp4-6.0.1/bin, so the phaser setup apparently has worked to some degree, but 
the log files (phaser.log and phasere.log) are missing (!!!) so I have no idea 
whether it has run cleanly. 
So the whole thing *may* be related to the setup now using the system-wide 
python installation instead of the ccp4-provided version (which is mandatory 
because the system does not like that one).
Since there are no log files giving more information I am stuck at this point.

Any suggestions?

[Please refrain from advising me to "use a different distribution" (and 
particularly from recommending SuSE), which is definitly not an option. 
Re-performing a lot of installations because of some stupid software problem is 
a nightmare for a system administrator.]

Regards
Oliver

  

---
 Dr. Oliver H. Weiergraeber
 Institute of Neurosciences and Biophysics
 Molecular Biophysics
 Research Centre Juelich
 D-52425 Juelich
 Germany
 Phone: +49-2461-612028
 Fax: +49-2461-612020
---

Re: [ccp4bb] exposing your Tev site

[Sebastien Violot]

Lukacs, Christine wrote:


Quick question about Tev sites –

We have a situation where our N-terminal TEV site is inaccessible to 
the protease. Looking at a close homolog, we realized that our first 
protein residue is already involved in a beta sheet. From peoples’ 
experience – how FEW residues can we add between the cleavage site and 
the first protein residue in order to make the cleavage site 
accessible to the protease for efficient cleavage of the tag? (Tagged 
protein behaves well but crystallizes poorly, we are hoping that 
cleavage of the tag will improve things)


Thanks for any anecdotes-

***Christine Lukacs*

Roche


---
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mensaje de correo no solicitado (SPAM), haz clic en el siguiente 
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---


Hi Christine,
According to our experience, a 3 amino-acids long linker (Ser-Gly-Ala) 
between TEV recognition sequence and your protein of interest is enough 
for efficient cleavage. Remember that after cleavage, this linker will 
be part of your protein to be crystallized.

Good luck,

Seb

--
Dr Sebastien VIOLOT
Dep. Biologia Estructural
Institut de Biologia Molecular de Barcelona, CSIC
Parc Científic de Barcelona
Josep Samitier 1-5
08028 Barcelona
Spain

Phone   +34 93 403 4957
Fax +34 93 403 4979
Email   [EMAIL PROTECTED]
URL www.ibmb.csic.es

Re: [ccp4bb] recommendation for heavy atoms used in lithium sulfate and ammonium sulfate

[artem]

Tiancen

If you have enough crystals to experiment with, just do a normal series of
heavy atom derivatives, starting with good old favorites such as mercury
and platinum. As an added touch of class you can try tungstate - it's like
sulphate, but has fat W so it can be used probably just like a halide
soak...

Let me know if you need more detailed help :)

Artem

> Dear all,
>
> Could any one recommend some heavy atoms used for crystals grown in 0.1M
> tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? I
> read from Hampton user guide of heavy atom kit that "high salt
> concentrations are not the ideal medium for heavy atom reactions with
> macromolecules", so is there any type of heavy atom we should avoid using?
> We do not have any experience in preparing heavy atom derivative, so any
> suggestions, experience or references will be greatly appreciated.
>
> Thanks in advance!
>
> Tiancen Hu
> Shanghai Institute of Materia Medica
>

Re: [ccp4bb] recommendation for heavy atoms used in lithium sulfate and ammonium sulfate

[Jeffrey . Kieft]

Tiancen,

How critical is it that the crystals remain in the sulfate salts?  I ask
because we recently had success with an RNA crystal that was very
intractable to heavy atom soaks because of very high lithium sulfate
growth conditions, and the fact that cryo-protection was best done by
increasing the lithium sulfate to near saturation.  At that high sulfate
concentration, many of our favorite heavy atoms (mostly divalent and
trivalent cations since the crystals were RNA) are insoluble, and the
high Li+ concentration competed off the heavy atoms.

What I did was move the crystals, in steps, from the 2M lithium sulfate
into high concentration of lithium Acetate.  What this did was increase
the solubility of the heavy atom cations, which still maintaining high
ionic strength and offering cryoprotection.  Ultimately, what worked was
3M lithium acetate + 100 mM cobalt hexammine or iridium hexammine (yes,
100 mM trivalent!).  We got a nice derivative, and in fact the
diffraction limit of the crystal increased by almost 0.5 Angstroms!  

This was with an RNA crystal, so your problems may be different, but
perhaps something analogous might work, or at least you can get some
ideas from our experience.  

In short, if solubility of the heavy atoms is an issue, you may be able
to move the crystals into an alternate high salt condition that will
give better solubility, and then crank the heavy atom concentration up
to compete with the salt.

Good luck,

Jeff

___
Jeffrey S. Kieft, Ph.D.
Assistant Professor
Dept. of Biochemistry and Molecular Genetics
University of Colorado School of Medicine
 
http://www.uchsc.edu/sm/bbgn/kieftj.htm
http://www.evolutionarygenomics.com/CERT/CERT.html
_
For mail:
UCHSC at Fitzsimons
Mail Stop 8101, PO Box 6511
Aurora, CO 80045
 
For courier/packages:
South Building RC-1, Room 9110
12801 East 17th Ave.
Aurora, CO 80010
 
phone:  303-724-3257
fax:  303-724-3215
email:   [EMAIL PROTECTED]
 
"Open your eyes.  You have only to see things clearly, to understand."
-Leonardo da Vinci

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Tiancen Hu
Sent: Thursday, April 19, 2007 10:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] recommendation for heavy atoms used in lithium sulfate
and ammonium sulfate

Dear all, 

Could any one recommend some heavy atoms used for crystals grown in 0.1M
tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate?
I read from Hampton user guide of heavy atom kit that "high salt
concentrations are not the ideal medium for heavy atom reactions with
macromolecules", so is there any type of heavy atom we should avoid
using? We do not have any experience in preparing heavy atom derivative,
so any suggestions, experience or references will be greatly
appreciated.

Thanks in advance!

Tiancen Hu
Shanghai Institute of Materia Medica

[ccp4bb] exposing your Tev site

[Lukacs, Christine]

Quick question about Tev sites - 
We have a situation where our N-terminal TEV site is inaccessible to the
protease.  Looking at a close homolog, we realized that our first
protein residue is already involved in a beta sheet.  From peoples'
experience - how FEW residues can we add between the cleavage site and
the first protein residue in order to make the cleavage site accessible
to the protease for efficient cleavage of the tag?  (Tagged protein
behaves well but crystallizes poorly, we are hoping that cleavage of the
tag will improve things)

Thanks for any anecdotes-

Christine Lukacs
Roche

Re: [ccp4bb] How to run SIGMAA twice in a row? (fwd)

[Peter Adrian Meyer]

> structure" running mode of SIGMAA. This is the run we really wanted to
combine
> the model phases with the MAD phases before going through further
density
> modifications with SOLOMON or DM.

I would have thought that you'd want to do this the other way around
(density modification on MAD before model phase combination) in order to
reduce possible model bias.

I'm curious...what's the reasoning for doing the model phase combination
first?

Pete


Pete Meyer
Fu Lab
BMCB grad student
Cornell University

Re: [ccp4bb] GUI problem with SIGMAA

[Huiying Li]

Hi Eleanor,

Thanks for doing the test. I did the same thing through run & view com 
option as Chris Rife suggested. SIGMMA did wonderful job once I was able 
to provide the second run with the correct FOM from the first round of 
MAD phase combination. The density quality (after SOLOMON) calculated with 
correct FOM is much better than the one with the FOMmad (the one I could 
only select through the GUI). With the much improved density we were able 
to fit in most of the side chains.


Thanks again for everyone who offered help.

Huiying


On Fri, 20 Apr 2007, Eleanor Dodson wrote:


I cant find any test data - will have to create it to test properly..

Eleanor

Have done so.. things are exactly asd you describe them
I ran the first job with 2 sets oF PHI and HL coeffs
Output PHCMBtest1 WCMBtest1 HLACMBtest1 etc

Then tried second and as you said there was no FOM allowed for the input 
"experimental" phase PHCMBtest1


It runs OK if I use the option run and view com file and insert the extra 
info into the LABI line


LABI FP= PHIBP=PHCMBtest1 etc and then I add WP=WCMBtest1

But that shouldnt be necessary as you say..
The only peculiarity I can see in the first SigmaA output is that the 
combined phases are in data set 0, but changing that doesnt help.


Any ideas GUI experts???

Eleanor

Huiying Li wrote:
Thank Eleanor and others for making suggestions. We have tested SIGMAA in 
CCP4i(1.4.4.1) again carefully. In the first run, we used "combine two sets 
of MIR phases" mode to combine 2 sets of MAD phases, and output column 
labels changed to PHCMBtest and WCMBtest. The second run was in "combine 
isomorphous phase with partial structure" mode. We had no problem assigning 
other column labels such as FP, PHIBP, ... because the pull-down menu of 
each label includes "list all labels" option. But FOM pull-down menu had no 
"list all labels" option, we could not select the label WCMBtest generated 
from the previous run. It seems to me there is a bug in the GUI that limits 
the option in FOM pull-down menu. It only occurs in the "combine 
isomorphous phase with partial structure" running mode of SIGMAA. This is 
the run we really wanted to combine the model phases with the MAD phases 
before going through further density modifications with SOLOMON or DM.


Regards,

Huiying

On Thu, 19 Apr 2007, Eleanor Dodson wrote:


Sorry about that

It is ages since I have used SIGMAA

The MAD recombnation gives you PHCMB WCMB HLAC HLBC etc
In that run I would make sure I renamed PHCMB WCMB PHCMBmadsWCMBmads or 
something
You might have to use the option Run and view com file, then edit in a 
line:

LABOUT PHCMB=PHCMBmads WCMB= etc

Then I think you may be able to do what you want - I suspect the poor 
program is getting lost between what is a default output label, and 
something already in the header.


Can you try that?
Or you use the Clipper utility to combine phases.. it is more mindless but 
you do have to convert your PHIC FOM to HLA HLB (HLC HLD ==0) using 
another clipper utility first

Eleanor



Huiying Li wrote:
My previous posting did not get answer, it might be a bit confusing. My 
questions is if I want to conbine phases from three different sources, 2 
MAD data sets and a partial model, can I run SIGMAA twice? Combining MAD 
phases first, and then add in the model phases to the pre-combined MAD 
phases. When I run SIGMAA the second time CCP4i GUI allows me to choose 
labels PHCMB, HLAC, HLBC, HLCC, HLDC, but doesn't let me use WCMB as the 
input FOM. Is this a bug in the GUI or SIGMAA simply does not allow to 
use WCMB as an input FOM?


Thanks in advance.

Huiying

-
Huiying Li, Ph. D
Department of Molecular Biology and Biochemistry
Natural Sciences I, Rm 2443
University of California at Irvine
Irvine, CA 92697, USA
Tel: 949-824-4322(or -1953); Fax: 949-824-3280
email: [EMAIL PROTECTED]
--










--
-
Huiying Li, Ph. D
Department of Molecular Biology and Biochemistry
Natural Sciences I, Rm 2443
University of California at Irvine
Irvine, CA 92697, USA
Tel: 949-824-4322(or -1953);  Fax: 949-824-3280
email: [EMAIL PROTECTED]
--

Re: [ccp4bb] How to run SIGMAA twice in a row? (fwd)

[Huiying Li]

Hi Pete,

The sigmaa-weighing scheme implemented in SigmaA routine is the very means 
to remove the potential model bias. Also, the model phases we used in the 
phase combination were simply from a backbone poly-Ala model generated 
from the best parts of the MAD-phased density (some of them from an 
ARP/wARP run) leaving out the surface regions where the map quality is 
marginal. Combining partial model phases with the experimental phases has 
been used as a way to improve the map quality while the partial model is 
still far from complete and most of  the side chains are not yet filled 
in. Once enough scattering mass has been built into the model and a 
reciprocal space refinement with CNS or REFMAC is warranted, the 
resulting 2Fo-Fc and Fo-Fc maps are often having much suprior quality for 
the further model building.


One drawback of combining partial model phases with the density-modified 
experimental phases is that one cannot run the density modification second 
time after the combination. I have not tested whether combining model 
phases with the density modified MAD phases produces good quality map 
(experts in the field can make comments). I did get significant 
improvement in the map quality with the combine-then-modify procedure.


HTH,

Huiying

On Fri, 20 Apr 2007, Peter Adrian Meyer wrote:


structure" running mode of SIGMAA. This is the run we really wanted to

combine

the model phases with the MAD phases before going through further

density

modifications with SOLOMON or DM.


I would have thought that you'd want to do this the other way around
(density modification on MAD before model phase combination) in order to
reduce possible model bias.

I'm curious...what's the reasoning for doing the model phase combination
first?

Pete


Pete Meyer
Fu Lab
BMCB grad student
Cornell University






--
-
Huiying Li, Ph. D
Department of Molecular Biology and Biochemistry
Natural Sciences I, Rm 2443
University of California at Irvine
Irvine, CA 92697, USA
Tel: 949-824-4322(or -1953);  Fax: 949-824-3280
email: [EMAIL PROTECTED]
--

[ccp4bb] Ecoli protein staining with AntiHis?

[Raji Edayathumangalam]

Hi Everyone,

I see a band that lights up in my anti-His Western blots.

While I investigate what else the band might be (truncated protein etc.), does 
anyone know whether
there is an E. coli protein that migrates ~ 30-40kDa (SDS-PAGE), which binds to 
anti-His antibodies?

Thanks.
Raji

[ccp4bb] Drug Target Crystallography and Structure Based Drug Discovery June 28-29 2007

[Bernhard Rupp]

Dear All:

A two day workshop on the cutting-edge developments in crystallography and
structure-based drug design will be held on June 28-29, 2007 in La Jolla
California. The workshop will be conducted jointly by myself and Dr. Ruben
Abagyan (Professor of Molecular Biology at The Scripps Research Institute
and Founder of Molsoft LLC).

The workshop entitled "Modern Drug Target Crystallography and Structure
Based Drug Design"
(http://www.ruppweb.org/workshops/Molsoft_workshop_2007.htm) is suitable for
executives, scientists, and active researchers in the field of biological
sciences and drug discovery, who wish to expand their knowledge in the
rapidly advancing field of high throughput drug target crystallography and
structure guided drug discovery. Includes live demonstrations of
crystallization (http://www.ruppweb.org/cryscam/cryscam_default.html),
practical structure solution, and ligand screening and drug discovery
software. 

PDF files of the presentations are provided a few weeks
ahead of the workshop for registered participants.

The workshop is designed for those who are considering using crystallography
and virtual ligand screening as a tool in their drug discovery research and
wish to get a concise overview of the techniques, as well as for researchers
already familiar with crystallography and in-silico, analysis, modeling and
docking, who wish to learn more about the latest developments in these
fields. 

For more information please visit: 
http://www.ruppweb.org/level1/Workshops.htm
E mail [EMAIL PROTECTED] or call (858) 625 2000 x108. 
Places are limited to 12 participants so please register early.
http://www.ruppweb.org/workshops/molsoft_training_reg.pdf

Thx, br
-
Bernhard Rupp
http://www.ruppweb.org/
-

Re: [ccp4bb] How to run SIGMAA twice in a row? (fwd)

[Peter Adrian Meyer]

Hi Huiying,

Thanks, I think I understand what your approach was.  I guess I was
overly-concerned about model bias during density modification of model
phases (I can explain to myself both why it's valid and why it's not; but
haven't been able to resolve the obvious contradiction there); although
with two mad phase sets in the mix as well it probably wouldn't have a
large effect in any event.

I'd also thought that sigma_a (and other weighting schemes) reduced model
bias, but that it wasn't possible to completely remove it.  It's been a
while since I read the sigma_a paper, thought.

But seeing as your map improved, then these apparently aren't significant
problems.

Thanks again,

Pete

> Hi Pete,
>
> The sigmaa-weighing scheme implemented in SigmaA routine is the very means
> to remove the potential model bias. Also, the model phases we used in the
> phase combination were simply from a backbone poly-Ala model generated
> from the best parts of the MAD-phased density (some of them from an
> ARP/wARP run) leaving out the surface regions where the map quality is
> marginal. Combining partial model phases with the experimental phases has
> been used as a way to improve the map quality while the partial model is
> still far from complete and most of  the side chains are not yet filled
> in. Once enough scattering mass has been built into the model and a
> reciprocal space refinement with CNS or REFMAC is warranted, the
> resulting 2Fo-Fc and Fo-Fc maps are often having much suprior quality for
> the further model building.
>
> One drawback of combining partial model phases with the density-modified
> experimental phases is that one cannot run the density modification second
> time after the combination. I have not tested whether combining model
> phases with the density modified MAD phases produces good quality map
> (experts in the field can make comments). I did get significant
> improvement in the map quality with the combine-then-modify procedure.
>
> HTH,
>
> Huiying
>
> On Fri, 20 Apr 2007, Peter Adrian Meyer wrote:
>
>>> structure" running mode of SIGMAA. This is the run we really wanted to
>> combine
>>> the model phases with the MAD phases before going through further
>> density
>>> modifications with SOLOMON or DM.
>>
>> I would have thought that you'd want to do this the other way around
>> (density modification on MAD before model phase combination) in order to
>> reduce possible model bias.
>>
>> I'm curious...what's the reasoning for doing the model phase combination
>> first?
>>
>> Pete
>>
>>
>> Pete Meyer
>> Fu Lab
>> BMCB grad student
>> Cornell University
>>
>>
>>
>>
>
> --
> -
> Huiying Li, Ph. D
> Department of Molecular Biology and Biochemistry
> Natural Sciences I, Rm 2443
> University of California at Irvine
> Irvine, CA 92697, USA
> Tel: 949-824-4322(or -1953);  Fax: 949-824-3280
> email: [EMAIL PROTECTED]
> --
>


Pete Meyer
Fu Lab
BMCB grad student
Cornell University

Re: [ccp4bb] Ecoli protein staining with AntiHis?

[Artem Evdokimov]

Depends on which antibody you're using. Different manufacturers tend to
light up different stuff. We very rarely see just one band - typically it's
a bunch of weak bands as well as smears.

 How strong is the band? Can you cut it out of the gel and do a tryptic
digest and MS identification? If the band is strong, consider the
possibility that you've somehow contaminated your strain with some other
expressor...

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Raji
Edayathumangalam
Sent: Friday, April 20, 2007 4:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ecoli protein staining with AntiHis?

Hi Everyone,

I see a band that lights up in my anti-His Western blots.

While I investigate what else the band might be (truncated protein etc.),
does anyone know whether
there is an E. coli protein that migrates ~ 30-40kDa (SDS-PAGE), which binds
to anti-His antibodies?

Thanks.
Raji