Re: [ccp4bb] ? steps after detwinning

[Seema Nath]

I've used phenix.xtriage and it showed that in all three cases i.e. (P31,P61 & 
P6122 space groups) "translational pseudosymmetry is very likely present). 
Again, when I used molrep in ccp4,it showed "translational pseudosymmetry is 
detected".

[ccp4bb] 2nd Oxford Symposium on Epigenetic Mechanisms in Health and Disease

[Frank von Delft]

Hi all, a symposium announcement:

The 2nd Oxford Symposium on Epigenetic Mechanisms in Health and Disease will be 
held 9-10th December 2010 in the Said Business School, Oxford.

The symposium aims to bring together clinical and preclinical scientists 
engaged at the forefront of control of inflammation and cancer through 
epigenetic and chromatin mechanisms.

Full details are here:http://www.thesgc.org/symposia/episymp2010/index.php

Cheers
phx

[ccp4bb] please disregard - test

[Bernhard Rupp (Hofkristallrat a.D.)]

posting test

BR

[ccp4bb] Chair in Structural Biology at The University of Auckland

[Shaun Lott]

The School of Biological Sciences at the University of Auckland, New Zealand invites applications for a Chair in Structural Biology. The ideal candidate will combine X-ray crystallography with a broad range of biophysical, biochemical and cell technologies to solve relevant biological problems of fundamental and applied nature. The successful candidate will establish his/her own research portfolio and actively contribute to teaching and graduate supervision. Commensurate with the seniority of the position, the appointee will also provide leadership for the Structural Biology Section within the School of Biological Sciences, which currently comprises five research groups and around 40 staff and students. 
 See http://www.bioscienceresearch.co.nz/research/structural-biology for details.  
Facilities currently available in the Structural Biology Laboratory include a Rigaku Micromax 007HF rotating anode generator and two Mar345 image plate detectors on a Dtb with mounting robot, with Osmic mirror optics and cryocooling; a Cartesian nanolitre dispensing robot for crystallization; numerous FPLC systems, and access to DLS, ITC, CD, high-field NMR (Bruker 600 MHz with cryoprobe), excellent cryo-EM (FEI Tecnai 12 and forthcoming Tecnai 20) and advanced mass spectrometry facilities.
The Laboratory is situated in the School of Biological Sciences, which has close to 200 staff and supervises over 150 graduate students. It also has strong links with the University of Auckland Medical School and forms part of the Maurice Wilkins Centre, a national Centre of Research Excellence. It is located on the University’s city campus which is in a beautiful park setting close to Auckland’s picturesque harbour.  
For general information on the position and informal enquiries, please contact Prof. Ted Baker (ted.ba...@auckland.ac.nz) 
  Applications close on 15 October 2010. To apply for this position and for further information and other conditions go to http://www.auckland.ac.nz/opportunities and search for position number 12305. 



The School of Biological Sciences at the University of Auckland, New Zealand 
invites applications for a Chair in Structural Biology. 

Re: [ccp4bb] Regarding quality of protein for crystallization

[Artem Evdokimov]

1)My protein has 14 cysteine residues.
>

That can be a big issue in itself :)


> 2)It is not a metal binding protein.
>

Are you sure, with this many Cys? Positive?


> 3)I have added the protease inhibitor cocktail + PMSF during sonication and
> cleavage. I saw only one band of protein bind on beads. Two band appears
> after cleavage.
>

Consider an alternative lysis method (detergents, etc.) - sonication can be
very un-gentle.


> 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs
> in Rosetta 2DE3
>

Nothing special here :)


> 5) I have to do MALDI and MS MS to know exact molecular weight and sequence
> that  differ  in both the proteins. Most of the  peaks in peptide mass
> fingerprint match exactly while some are at different position and
> size. Amino acids sequence similarity  in both the cases are upto 1-405
> amino acids (as per limitation of peptide mass fingerprint it show some
> region upto 1-405 matching in both the proteins).
>

My bet is either on an incompletely reduced S-S or on PTM.


>
> I  have certain doubt/solution(s) on the basis of yours feedback and above
> information.
>
> 1) How come PTM can play role when protein expressed in bacteria.
>

PTM is present in pretty much any organism. For example, peptide deformylase
and methionine aminopeptidase process the N-terminus of bacterially
expressed proteins - depending on amino acid in position 2. That's PTM.
Ribosomal (and some other) proteins can be acetylated in E. coli. There's
one biotinylated protein (OK, that one's special, I admit) and of course
there's acyl carrier. Proteins can be poly-hydroxybutyrated in E. coli
although that latter modification can go away during purification. Proteins
can be deaminated and methylated, and so on and so forth. And that's before
we go into membrane anchoring modifications, or glycosylation such as the
one mentioned earlier or this one (
http://jb.asm.org/cgi/content/full/188/5/1798) and of course there's
non-enzymatic glycosylation such as one suggested here (
http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2001.02304.x/pdf).


> 2) TEV is a very specific protease hence non specific cleavage less likely
> occur.
>

Nearly never. Your sequence either has a TEV-like target site or it doesn't.
Usually the latter.


> 3) Cysteine residues or temperature dependent expression may be one of the
> reason of two band of protein.
>


>
> Further suggestion to get rid of this problem will be highly appreciated.
>

Express in another system, or systematically try out several commercial
strains of E. coli... find the pesky modification site and eliminate it...

Artem

>
>
>  With Regards
>
> *Vikrant
> *
>
>
>

[ccp4bb] trouble shooting

[Ray Brown]

Hi CCP4BB folks,
 
I am a freelance protein crystallographer looking to offer advice, 
problem-solving (protein purification, crystallization, X-ray data collection, 
crystallographic computing and model building etc), analysis of structure-based 
projects or provide hands-on assistance. 

 
Based in Boston USA, but I am willing to travel. Looking for temporary, ‘as 
needed’, ad hoc, contract, intermittent or short-term jobs.
 
I have successfully done X-ray crystallography of RNA, chromatin, protein 
complexes, membrane proteins and also for structure-guided drug discovery.
 
If you need someone with wide practical, bench top experience to help with a 
backlog or trouble shoot crystallography projects then please contact me.
 
Ray Brown, PhD.
 
Email : ray-br...@att.net

Re: [ccp4bb] problem in heavy metal soaking

[vincent Chaptal]

Hi,
to remove 'some of the voodoo' out of it, we recently published a paper
showing a really fast_and_easy (and cheap) technique to check HA binding
on cysteines.
One of the most interesting result was that we could see binding of
mercury and gold on 1 protein, but not platinum. We didn't try to
explain why, but this result tells you not to use platinum on this
particular protein... Then it's up to you to use pre-labelling or
soaking, but you should avoid platinum to incorporate the HA (in this
particular case again).
In another case, we showed that some platinum were better than other to
yield complete labelling.
This method (FD-HAL) isn't the ultimate answer to phasing but removes
some mystery, and will save you time on the beam line by working on
samples that you know have incorporated HA.
http://www.ncbi.nlm.nih.gov/pubmed/20152903
best
vincent

Paul Smith wrote:

Hi Seema,

In theory, Ammonium Sulfate contains some small fraction of neutral ammonia 
which can act as a strong nucleophile and react with many heavy metal compounds.

That being said, I recently phased two structures with mercury soaks, both of 
which contained ammonium sulfate.  The first was a thimerisol soak with 2M 
AmSO4 in the mother liquor, and the second with a MeHgCl soak with 0.2M AmSO4.  
Both were fairly routine.

I think others will agree that with heavy metals, logic and theory can go right 
out the window.  There is no way of knowing if it will work, you just have to 
try.

One tip I can offer is to use fresh stocks of metals dissolved at saturating 
concentrations in water and used on the same day.  The fresher the better in my 
experience, but it could just be voodoo.

Good luck!

--Paul

--- On Sun, 9/26/10, Seema Nath  wrote:



From: Seema Nath 
Subject: [ccp4bb] problem in heavy metal soaking
To: CCP4BB@JISCMAIL.AC.UK
Date: Sunday, September 26, 2010, 9:00 AM
I'm working with a protein which
crystallizes in a mixture of PEG6K with 0.2M AmSO4,my
question is if there's any problem if I want to soak heavy
metal derivatives in this crystallizing condition? Does
AmSO4 interfere in heavy-metal soaking ? if yes, what's the
reason?
Thank you in advance.








--

Vincent Chaptal

Dept. of Physiology at UCLA

http://www.physiology.ucla.edu/Labs/Abramson/index.html


Phone: 1-310-206-1399


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[ccp4bb] Ab Biacore

[dhurjati putcha]

Dear all,
Sorry about non crystallographic query. Are there any binding studies (using
Biacore) on interactions between antibody and anti-antibody (particularly
mouse antibody)?
thank you
Putcha

Re: [ccp4bb] protein turns brown

[Guenter Fritz]

Sandy,
like mentioned previously, sounds like FeS.
record an optical spectrum. Or even better, check whether there is 
somebody on the campus running an EPR machine (equipped for helium 
temperature measurements)
Maybe  a new FeS  protein? FeS is not necessarily required as redox 
cofactor. It can have any other function . See e.g. primase from 
eukaryotes (recent structures)
Or in your native (maybe eukarytic) protein is a Zn.  E.coli places 
sometimes a FeS instead of a Zn in the site.
Less likely but further possibility: Ni2+ bound to the protein. If DTT 
is added: Ni2+ -S coordinated is not redox stable and oxidizes to Ni3+ 
(brownish colour).

HTH
Guenter

Dear all,

I have purified protein from E.coli. expression system. the protein 
has been purified with three independant columns. Now during 
concentration step using amicon, the protein shows brown colour. what 
could be the reason.


best regards and Thanks,
sandy
 

Re: [ccp4bb] Home Source Options

[Roger Rowlett]

  
  
Paul,
  
  We've been running an Oxford Diffraction (now Agilent) Gemini
  system for nearly two years now. We have the older PX Ultra source
  for protein, and a Ruby (135 mm) detector. As an educational
  institution, we actually do a lot of structure solution in-house,
  both for convenience and for instructional purposes. The newer
  Supernova source is (I think) about 4X brighter than our source,
  and the units are smaller and consume less power as well. The
  software, CrysalisPro, is very good at integration and scaling,
  and interfaces well with CCP4.
  
  We are running our sources at about 30% of the available time, and
  are still acceptably strong after 18+ months. We anticipate
  needing to replace them at our current usage rate every 24-36
  months, or when they fail. They are about $3000 each at last
  check. Cooling is 2L/min of tap water, power supply is nothing
  special, just 240V and 120V supplies, 15-25A, if I recall
  correctly. I think the newer sources may have less in the way of
  power requirements.
  
  The only maintenance issues we have had is with the cooling
  plumbing: we replaced a couple of solenoid valves, a pump, and a
  flow meter due to clogging/sticking, but I think most of these
  issues may have originated in the factory, not during operation.
  The electronics seem to be pretty robust. We are replacing the
  brass main cooling pump fairly frequently, but they are not very
  expensive, and are available from a third party vendor. The
  biggest expense has been the cryogens for the cryojet.
  
  We did quite a bit of research before writing a successful
  proposal for our XRD instrument. At least as of 2 years ago, we
  couldn't afford to purchase or operate any other instrument in a
  small college environment. Rotating anode sources are out of the
  question in terms of maintenance and cost of operation for us. The
  sealed tube sources are the way to go, and Oxford was the first to
  offer a sealed tube instrument with capability similar or
  equivalent to rotating anode sources. The data I can get out of
  the PX Ultra/Ruby CCD is similar to that of a Rigaku RU-200 +
  RAXIS-IV system I used at the NIH. We spent a little over $400K
  for the entire system 2 years ago. Maintenance has been minimal in
  terms of cost, even if we totaled up the cost of parts we replaced
  under warranty. For $12K/yr (last time it was offered to me),
  Oxford/Agilent will give you 100% maintenance, which is not a bad
  deal if you don't want to deal with maintenance yourself.
  
  Cheers, Roger Rowlett
  

On 9/26/2010 10:06 PM, Paul Smith wrote:

  I'm interested in opinions/advice on home source systems.

As synchrotron beamlines are more powerful and more accessible than ever, a home source is really only needed for crystal screening, if at all.

With that idea in mind, what are options out there for buying and running a home source in the most cost friendly way possible?

I'm aware of most of the options from the big players (Rigaku, Oxford, Bruker), but I would like input on which setups cost the least to buy, run year in and year out, and require the least in terms of facilities setup (cooling water, power supply, etc.)

Any positive/negative experiences worth sharing?

Thanks,

--Paul


  

Re: [ccp4bb] Regarding quality of protein for crystallization

[Pius Padayatti]

ealrier on this forum Artem have posted about PTM
in bacteria.
this answer your question on PTM in bacteria.


QUOTE

"Yes, this does happen.
Spontaneous α-N-6-Phosphogluconoylation of a "His Tag" inEscherichia
coli:The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45N4K22-R&_us
er=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C50221&_version=1&
_urlVersion=0&_userid=10&md5=453fd46805ef7137c62705a5ae80384e

There are other options out there too but this one comes to mind first.

Artem"

On Mon, Sep 27, 2010 at 9:09 AM, vikrant saa  wrote:
> Dear all
> Thanks for yours valuable suggestion.
> Just some addition information to make the query clear.
>
> 1)My protein has 14 cysteine residues.
> 2)It is not a metal binding protein.
> 3)I have added the protease inhibitor cocktail + PMSF during sonication and
> cleavage. I saw only one band of protein bind on beads. Two band appears
> after cleavage.
> 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in
> Rosetta 2DE3
> 5) I have to do MALDI and MS MS to know exact molecular weight and sequence
> that  differ  in both the proteins. Most of the  peaks in peptide mass
> fingerprint match exactly while some are at different position and
> size. Amino acids sequence similarity  in both the cases are upto 1-405
> amino acids (as per limitation of peptide mass fingerprint it show some
> region upto 1-405 matching in both the proteins).
>
>
> I  have certain doubt/solution(s) on the basis of yours feedback and above
> information.
>
> 1) How come PTM can play role when protein expressed in bacteria.
> 2) TEV is a very specific protease hence non specific cleavage less likely
> occur.
> 3) Cysteine residues or temperature dependent expression may be one of the
> reason of two band of protein.
>
> Further suggestion to get rid of this problem will be highly appreciated.
>
>
>  With Regards
>
> Vikrant
>
>



-- 
Pius S Padayatti
Scientist,
Polgenix, Inc.
11000 Cedar Ave, Suite 260
Cleveland, OH 44106
Phone: 216-658-4528
Fax: 216-658-4529

Re: [ccp4bb] Regarding quality of protein for crystallization

[Jürgen Bosch]

How about incomplete TEV cleavage ?
Your protein is a dimer/multimer in solution and some of it is cleaved and some 
not and they stick together and are separated on the SDS gel ?

Jürgen

-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Sep 27, 2010, at 9:09 AM, vikrant saa wrote:

> Dear all
> Thanks for yours valuable suggestion.
> Just some addition information to make the query clear.
>  
> 1)My protein has 14 cysteine residues.
> 2)It is not a metal binding protein.
> 3)I have added the protease inhibitor cocktail + PMSF during sonication and 
> cleavage. I saw only one band of protein bind on beads. Two band appears 
> after cleavage.
> 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in 
> Rosetta 2DE3
> 5) I have to do MALDI and MS MS to know exact molecular weight and sequence 
> that  differ  in both the proteins. Most of the  peaks in peptide mass 
> fingerprint match exactly while some are at different position and size. 
> Amino acids sequence similarity  in both the cases are upto 1-405 amino acids 
> (as per limitation of peptide mass fingerprint it show some region upto 1-405 
> matching in both the proteins).
>  
>  
> I  have certain doubt/solution(s) on the basis of yours feedback and above 
> information.
>  
> 1) How come PTM can play role when protein expressed in bacteria.
> 2) TEV is a very specific protease hence non specific cleavage less likely 
> occur.
> 3) Cysteine residues or temperature dependent expression may be one of the 
> reason of two band of protein.
>  
> Further suggestion to get rid of this problem will be highly appreciated.
>  
>  
>  With Regards
>  
> Vikrant
>  
> 
> 

[ccp4bb] Regarding quality of protein for crystallization

[vikrant saa]

Dear all 
Thanks for yours valuable suggestion.
Just some addition information to make the query clear.

1)My protein has 14 cysteine residues. 
2)It is not a metal binding protein.
3)I have added the protease inhibitor cocktail + PMSF during sonication and 
cleavage. I saw only one band of protein bind on beads. Two band appears after 
cleavage.
4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in 
Rosetta 2DE3
5) I have to do MALDI and MS MS to know exact molecular weight and sequence 
that  differ  in both the proteins. Most of the  peaks in peptide mass 
fingerprint match exactly while some are at different position and 
size. Amino acids sequence similarity  in both the cases are upto 1-405 amino 
acids (as per limitation of peptide mass fingerprint it show some region upto 
1-405 matching in both the proteins).


I  have certain doubt/solution(s) on the basis of yours feedback and above 
information.

1) How come PTM can play role when protein expressed in bacteria.
2) TEV is a very specific protease hence non specific cleavage less likely 
occur.
3) Cysteine residues or temperature dependent expression may be one of the 
reason of two band of protein. 


Further suggestion to get rid of this problem will be highly appreciated.

 
 With Regards
 
Vikrant

Re: [ccp4bb] ? steps after detwinning

[Eleanor Dodson]

Do you have translational pseudo symmetry - what is the evidence?
It can confuse space group assignment..
Eleanor

Seema Nath wrote:

After running phenix.xtriage the possible point group is P622 and possible spacegroups 
are P622,P6122,P6522,P6222,P6422,P6322. Using this information when I run PHASER, P6322 
is shown to be the most probable one.After 5 cycles of phenix.refine R-& R-free - 
57 & 60 respectively.
From other crystallization papers, I found that data having translational 
pseudosymmetry had been solved in low-symmetry spacegroup firstly and then in 
higher symmetry.My query is:Is the data in lower symmetry spacegroup used  for 
higher symmetry or the raw-data is processed individually in higher symmetry? if 
raw data is used,then why not starting with higher symmetry directly? if data got 
in lower symmetry group is used,then what is the procedure?Are the .mtz & .pdb 
files generated in Refmac used for higher symmetry?
Thanks in advance. 

Re: [ccp4bb] ? steps after detwinning

[Seema Nath]

After running phenix.xtriage the possible point group is P622 and possible 
spacegroups are P622,P6122,P6522,P6222,P6422,P6322. Using this information when 
I run PHASER, P6322 is shown to be the most probable one.After 5 cycles of 
phenix.refine R-& R-free - 57 & 60 respectively.
From other crystallization papers, I found that data having translational 
pseudosymmetry had been solved in low-symmetry spacegroup firstly and then in 
higher symmetry.My query is:Is the data in lower symmetry spacegroup used  for 
higher symmetry or the raw-data is processed individually in higher symmetry? 
if raw data is used,then why not starting with higher symmetry directly? if 
data got in lower symmetry group is used,then what is the procedure?Are the 
.mtz & .pdb files generated in Refmac used for higher symmetry?
Thanks in advance.

[ccp4bb] Postdocs on Hsp90 in Sussex

[Laurence Pearl]

2 Postdoctoral Research Fellows in
Structural Biology of Hsp90 complexes (Ref 041)

Genome Damage and Stability Centre
School of Life Sciences
University of Sussex, Brighton, U.K.

Two Wellcome Trust-funded Postdoctoral positions are available immediately in 
the laboratory of Professor Laurence Pearl FRS to study the structural basis 
for client protein recruitment and activation by the Hsp90 molecular chaperone 
system. The School of Life Sciences is very well equipped for all aspects of 
modern structural biology, with state-of-the-art laboratories for molecular 
biology, recombinant expression in bacterial and eukaryotic systems, 
biochemistry, biophysics and X-ray crystallography. Excellent synchrotron 
access (~ 2 days/month) is available through rolling beam allocation programmes 
at Diamond and ESRF.

Applicants must have a PhD, and experience in recombinant expression and 
protein purification. Experience of crystallisation and X-ray crystallography 
would be an advantage. Information on our previous work in this field can be 
found by searching PubMed (pearl [au] and hsp90).

The postholders will be responsible for expression, purification, 
crystallization and structural analysis of protein complexes involving Hsp90, 
co-chaperones and client proteins that depend on Hsp90 for their biological 
function.

Both posts are full time and funded until 30 Sept 2011 in the first instance. 
Salary: £29,853 - £35,646 per annum.

Informal enquiries can be made to Professor Pearl laurence.pe...@sussex.ac.uk

Further Particulars and Application Forms can be found at : 
http://www.sussex.ac.uk/jobs/

Closing date: 10 October 2010


Laurence H. Pearl PhD FMedSci FRS

Professor of Structural Biology and Head of School of Life Sciences
University of Sussex, Brighton, BN1 9RH, UK

Phone +44-(0)1273 876 544: PA +44-(0)1273 872 699 

"Live Modestly and do Serious Things .. "
- Dorothy Crowfoot Hodgkin