[ccp4bb] Job Opportunities at UC Merced - Postdoctoral Scholar and Junior Specialist positions

2020-11-10 Thread Michael Thompson
Dear CCP4BB,

The Thompson Lab (https://thompsonlab.science) at UC Merced is recruiting a 
Postdoctoral Scholar and a Junior Research Specialist:

The Postdoc will work on projects related to multi-temperature and 
time-resolved (T-jump) crystallography at synchrotrons and XFELs. Applicants 
with experience in crystallography and/or data analysis are especially 
encouraged to apply. Job posting with additional details here:
https://aprecruit.ucmerced.edu/JPF01028

The Junior Specialist will support wet lab operations and assist with a variety 
of research projects in structural biology. Job posting here:
https://aprecruit.ucmerced.edu/JPF01024

The Thompson Lab is a new lab, and members will have an outsized role in 
shaping the lab culture. We're also moving into brand new lab space this 
winter. Our lab has regular opportunities for synchrotron beamtime, and we are 
competitive for XFEL beamtime (recently we were one of a handful of groups 
invited to submit a proposal for a scientific campaign at LCLS).

UC Merced is the youngest of the Universities of California, admitting its 
first students in 2005. In 15 years, the University has grown to almost 10,000 
students and has a vibrant and extremely diverse campus. There are mostly young 
faculty, and the university is pushing to rapidly expand its research 
reputation, making a lot of investments in infrastructure. With access to all 
the UC resources, and a rapid pace of growth, UC Merced is a really exciting 
place to be.

Merced is a great place to live. It is about 1 hour from the gates of Yosemite 
National Park, and a short 2-hour drive to San Francisco. (You can pack your 
samples in a car and drive to 2 synchrotrons and an XFEL!!) There's also a nice 
downtown area in Merced with good restaurants and entertainment, and it is one 
of the few remaining places in California where the cost of living is still 
relatively low (lowest cost of living for any UC campus).

Interested applicants should apply using the links above and reach out to 
mthompso...@ucmerced.edu. Please forward to anyone who you think might be 
interested.

Thanks!

Mike Thompson

---
Michael C. Thompson, Ph.D.
(he/him/his)
Assistant Professor
Dept. of Chemistry & Chemical Biology
University of California, Merced
https://thompsonlab.science



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[ccp4bb] random half data sets

2014-08-12 Thread Michael Thompson
Hello All,

I'm interested in doing some comparisons of random half data sets, inspired by 
statistics like CC1/2, CC*, etc. Does CCP4 contain some tool to split unmerged 
data into 2 random sets? Or is there some way to intercept this information 
from something like XSCALE or Aimless, etc.? Thought I would ask before trying 
to write my own script.

Thanks,

Mike








-- 
Michael C. Thompson

Postdoctoral Scholar

Institute of Genomics and Proteomics

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Off Topic- Fe-S electron transfer

2013-10-01 Thread Michael Thompson
Maybe there is a reason you haven't tried this, but can you just mutate the 
cysteines that ligate the cluster? Serine would be a nice choice.

Mike






- Original Message -
From: Yuri Pompeu yuri.pom...@ufl.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, October 1, 2013 11:33:50 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Off Topic- Fe-S electron transfer

Dear Community, 

I apologize for the off topic question but it is hard to resist since so many 
of our colleagues are incredibly knowledgeable.

I am looking for a way to block or even remove the 2Fe-2S cluster in my enzyme. 
This cluster is involved in transferring electron from the Alpha to the Beta 
subunit and I wish to block this for an experiment. 
Does anyone know of a reagent or procedure that is known to do this (elegance 
is optional)? I first thought of EDTA but I don't think it will work given the 
covalent character of the Fe-S bonds.

Thank you

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] off topic: good peak on gel filtration

2013-06-29 Thread Michael Thompson
Hi Peter,

I won't claim to be an expert, however, there is a small textbook by Robert K. 
Scopes called Protein Purification: Principles and Practice, that has very 
good practical and theoretical information about column chromatography (among 
other methods). I have found this book useful a number of times when I have had 
similar questions.

Good luck,

Mike







- Original Message -
From: Peter Hsu hsuu...@u.washington.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Saturday, June 29, 2013 2:01:15 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] off topic: good peak on gel filtration

Hi all,

I've generally always thought as long as the peak was symmetrical and not too 
broad would suggest a good sample. However, looking at my previous runs in the 
past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader 
peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on 
the columns). I'm curious to see what people's views are as far as what 
constitutes a broad peak and how much that can end up affecting crystallization 
of the sample. 

Thanks for any responses.

Peter

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] gelification of a pure protein

2013-04-22 Thread Michael Thompson
Does your protein have multiple exposed Cys residues? I have observed this 
before with a protein I worked with that had many exposed Cys residues. In my 
case I could add more DTT and minutes later the gel would be completely 
dissipated. My hand-wavy explanation was that the protein stays folded but 
becomes highly crosslinked when concentrated and exposed to air. I did have 
some small amount of DTT in my buffer to start with, but I assume it became 
oxidized relatively quickly (as DTT does), leaving my protein thiols to go wild 
and make S-S bonds between themselves. 

Good luck,

Mike






- Original Message -
From: Pascal Egea pas...@msg.ucsf.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, April 22, 2013 3:36:24 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] gelification of a pure protein


Dear All, 


I am presently faced with a peculiar case in the lab. We are expressing a 
protein in E. coli and we are able to express it as a fusion protein without 
problems . Fusion cleavage goes well and the final product looks homogenous by 
size-exclusion chromatography with the expected molecular weight. There are no 
signs of aggregation. However when we lower the salt concentration by dialysis 
then the protein forms a gel. transparent , optically clear, with no fluffy 
material (in the cold room). 


Gelification seems to occur when we lower the concentration below 100 mM NaCl. 
This protein has a fairly high pI (~9.0). Attempts to reverse the process by 
gentle heating or salt addition have been so far unsuccessful. It is not a 
thermophilic protein. We have not been able to obtain crystals so far. 



Has anyone already observed this kind of behavior and/or have any suggestions? 


Many thanks in advance . 

-- 
Pascal F. Egea, PhD 
Assistant Professor 
UCLA, David Geffen School of Medicine 
Department of Biological Chemistry 
Boyer Hall room 356 

611 Charles E Young Drive East 
Los Angeles CA 90095 
office (310)-983-3515 
lab (310)-983-3516 
email pe...@mednet.ucla.edu 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] space group determination problem

2013-03-15 Thread Michael Thompson
As a follow up to Ian's suggestion, the LABELIT software (sorry for a non-CCP4 
suggestion) will create artificial precession images from your raw oscillation 
images. 

Documentation can be found here:

http://adder.lbl.gov/labelit/

And an article describing the functionality can be found here:

http://cci.lbl.gov/publications/download/CCN_2011_p15.pdf

Hope it helps,

Mike







- Original Message -
From: Ian Tickle ianj...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, March 15, 2013 8:51:47 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] space group determination problem






Hi Gengxiang, 

Personally I find it impossible to reliably assign a space group from 
integrated reflections because you just don't know if the apparent systematic 
absence violations are due to a TDS streak or overlapping neighbouring strong 
spots. In the old days (i.e. when we had precession cameras) we would never 
do this: we would look at the images and see if there was actually a spot at 
the Bragg position. Now technology has advanced and with rotation images it's 
much harder to do this. Maybe it's possible to make pseudo-precession images? 

What I would do is assume the worst and assign it temporarily as P422; then let 
the HA or MR program sort out the space group by trying all the possibilities; 
it's only CPU time after all! 


My 2p's worth. 

-- Ian 




On 15 March 2013 15:09, gengxiang zhao  gzh...@gmail.com  wrote: 


Dear CCP4s, 

I am looking for more experienced concerns to determine which space group my 
crystal is. At present, we take it as P42212 (#94). 

HKL is below: 

Intensities of systematic absences 
h k l Intensity Sigma I/Sigma 

0 0 9 -58.6 40.8 -1.4 
0 0 11 -204.4 53.9 -3.8 
0 0 13 -57.1 62.8 -0.9 
0 0 15 -470.6 92.7 -5.1 
0 0 17 -626.1 105.1 -6.0 
0 0 19 -64.7 62.4 -1.0 
0 0 21 266.6 75.9 3.5 
0 0 23 1372.4 116.4 11.8 
0 0 25 -543.9 84.8 -6.4 
0 0 27 -396.8 93.1 -4.3 
0 0 29 -598.8 102.1 -5.9 
0 0 31 617.4 116.2 5.3 
0 0 33 445.4 93.8 4.7 
0 0 35 -64.5 89.5 -0.7 
7 0 0 -241.4 134.7 -1.8 
9 0 0 -375.8 55.5 -6.8 
11 0 0 -39.1 61.8 -0.6 
13 0 0 -356.1 78.1 -4.6 
15 0 0 -262.6 65.6 -4.0 
17 0 0 -324.7 89.3 -3.6 
19 0 0 -178.7 88.5 -2.0 
21 0 0 -726.3 115.3 -6.3 
23 0 0 -189.4 131.0 -1.4 
25 0 0 157.7 157.5 1.0 
27 0 0 -591.5 213.4 -2.8 
29 0 0 -111.7 198.4 -0.6 
31 0 0 -94.2 247.0 -0.4 
33 0 0 -169.8 306.5 -0.6 
35 0 0 -71.2 347.8 -0.2 
39 0 0 -82.8 417.9 -0.2 


Thanks a lot. 
Best Wishes, 
Gengxiang 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] disulfide engineering

2013-02-28 Thread Michael Thompson
Adding 1-10mM copper sulfate is often a good way to oxidize disulfide bonds, 
although some proteins cannot tolerate this treatment.

Mike


- Original Message -
From: Jacob Keller j-kell...@fsm.northwestern.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, February 28, 2013 3:09:18 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] disulfide engineering

Along these lines, what reagents do people use to promote disuflide bonds, 
i.e., the anti-DTT? 


JPK 


On Thu, Feb 28, 2013 at 2:06 AM, David Briggs  drdavidcbri...@gmail.com  
wrote: 




You might want to try Disulfide by design 

http://cptweb.cpt.wayne.edu/DbD2/ 

Cheers 

Dave 


On Feb 28, 2013 6:55 AM, Careina Edgooms  careinaedgo...@yahoo.com  wrote: 





Dear CCP4 members 


I wish to engineer a disulfide bond at the dimer interface of a protein I am 
working with. Does anyone know of any available software to assist with this? 


Best 
Careina 



-- 
*** 
Jacob Pearson Keller, PhD 
Postdoctoral Associate 
HHMI Janelia Farms Research Campus 
email: j-kell...@northwestern.edu 
*** 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] need some suggestions for crystallization

2013-02-04 Thread Michael Thompson
Hi Dave,

ProteinaseK is also a good one. Crystallizes rapidly, big crystals, and 
relatively high resolution data (1.0-1.5A) usually. You can also buy the 
lyophilized powder from sigma and prepare the sample directly from the 
commercial material. We use proK for a course here at UCLA, so if you are 
interested I can send you more details about the protocol.

Mike



- Original Message -
From: David Roberts drobe...@depauw.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, February 4, 2013 8:03:09 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] need some suggestions for crystallization

So, I know I say this every time I post on this board, but here it goes 
again.

I'm at an undergrad only school, and every 2 years I teach a class in 
protein crystallography.  This year I'm being super ambitious, and I'm 
going to take a class of 16 to the synchrotron for data collection.  
It's just an 8 hour thing, to show them the entire process.  I'm hoping 
that we can collect 5-6 good data sets while there.

I would like them to grow their own crystals, and go collect data. Then 
we'd come back and actually do a molecular replacement (pretty 
easy/standard really).  Just to get a feel for how it works.

The protein I do research on is not one that I would push on this, as 
the crystals are hard to grow, they are very soft, and the data just 
isn't the best (resolution issues).  I do have a few that will work on 
my proteins, but I was thinking of having others in the class grow up 
classic proteins for data collection.  Obviously lysozyme is one, but I 
was wondering what other standard bulletproof conditions are out there.

Can you all suggest some protein crystallization conditions (along with 
cryo conditions) for some commercially available proteins?  I'm looking 
to get 6-8 different ones (and we'll just take them and see how it 
goes).  I wouldn't mind knowing unit cell parameters as well (just a 
citation works, I can have them figure it out).  I have about 7 weeks to 
get everything grown and frozen and ready to go.

Any help would be greatly appreciated.  It always amazes me how helpful 
this group is.  Thank you very much.

Dave

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Fwd: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Michael Thompson
While neither of these references detail the development of protein 
crystallography, they are excellent stories of its birth:

1.) A book written by Richard Dickerson, Present at the flood

2.) A recent review in JMB by Strandberg, Dickerson, and Rossmann: 50 years of 
Protein Structure Analysis

We are lucky to have Richard Dickerson as emeritus faculty here at UCLA, 
because he cares very much for the history of science. Although I do not have a 
personal relationship with him, I always enjoy the opportunity to hear him talk 
about the beginnings. A couple years ago, we had a symposium to celebrate the 
50th anniversary of the first protein structures with guest speakers including 
Richard Dickerson, David Davies, Brian Matthews, Michael Rossmann, and Bob 
Stroud. Surprisingly, I cannot google my way to a recording of the lectures. 
I'm sure someone got a video or at least an audio recording, so if I can find 
it I will post a link.

Mike T








- Original Message -
From: Jim Pflugrath jim.pflugr...@rigaku.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, June 6, 2012 12:31:56 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Fwd: [ccp4bb] Fun Question - Is multiple isomorphous 
replacement an obsolete technique?


And for more Personal Reflections, one may wish to take a gander at the Rigaku 
Webinar series with presentations by Brian Matthews and Michael G. Rossmann. 


Jim 







From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Carter, Charlie 
[car...@med.unc.edu] 
Sent: Wednesday, June 06, 2012 2:05 PM 
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Fwd: [ccp4bb] Fun Question - Is multiple isomorphous 
replacement an obsolete technique? 







Begin forwarded message: 



Date: June 6, 2012 3:05:16 PM EDT 

To: aaleshin  aales...@burnham.org  

Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an 
obsolete technique? 


There are four such papers in Methods in Enzymology, Vols 368 and 374: 

David Blow: How Bijvoet Made the Difference: The Growing Power of Anomalous 
Scattering V. 374, pp. 3-22 

Brian Matthews: Transformations in Structural Biology: A Personal View V. 368 
pp. 3-10 

Michael Rossmann: Origins V. 368, pp. 11-21 

Ulrich W. Arndt: Personal X-ray Reflections V. 368, pp. 21-45 

These reminiscences are there entirely because my co-Editor Bob Sweet felt 
exactly the same way Alex does. 

Charlie 

On Jun 6, 2012, at 2:12 PM, aaleshin wrote: 



I wonder if anyone attempted to write a historic book on development of 
crystallography. That generation of crystallographers is leaving this world and 
soon nobody will be able to say how the protein and non-protein structures were 
solved in those days. 





Alex 
... 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Matthews coeff. from model

2012-03-12 Thread Michael Thompson
Is there a difference in the psv between DNA and RNA? I assumed (possibly 
incorrectly) that they would be very close if not exactly the same? Is mattprob 
more applicable to protein-DNA complexes than Protein-RNA complexes?

Thanks for the insight,

Mike



- Original Message -
From: Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com
To: Michael Thompson mi...@chem.ucla.edu, CCP4BB@JISCMAIL.AC.UK
Sent: Monday, March 12, 2012 12:48:42 PM GMT -08:00 US/Canada Pacific
Subject: RE: [ccp4bb] Matthews coeff. from model

 I can't imagine the results would be very different for protein-DNA vs. 
 protein-RNA.

The reason protein-nucleic acids is an extra category in mattprob is largely 
due to poorer statistics 
resulting from limited sample size and hence no reliable resolution dependence 
can be computed.

In addition the partial specific volumes for protein (0.74 cm3/g) and nucleic 
acids (0.50 cm3/g) are
different, so an exact calculation needs to consider their ratio to obtain the 
correct psv estimate  

BR

- Original Message -
From: Tim Gruene t...@shelx.uni-ac.gwdg.de
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, March 12, 2012 11:07:29 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Matthews coeff. from model

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear James,

I do not know such a tool, but you can use 140A^3/a.a. and 380A^3/base to 
calculate the solvent content by hand.

Regards,
Tim

On 03/12/2012 06:35 PM, james09 pruza wrote:
 Dear CCP4bbers,

 Is there any tool to calculate the Matthews coefficient from a 
 crystallographic model of RNA-protein complex?

 Thanking you.
 James.

 

- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A
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--
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Protein Fold Motifs- off-topic

2012-01-20 Thread Michael Thompson
In addition to the book recommended by Jeff, another good one is Protein 
Structure and Function by Petsko and Ringe. A bit less cumbersome than Branden 
and Tooze, but not as complete (it's a relatively thin paperback, rather than a 
full-blown textbook). If you just want a nice protein domain cheat sheet, it 
should be adequate.

Mike



- Original Message -
From: Jeff Headd jjhe...@lbl.gov
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, January 20, 2012 10:37:51 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Protein Fold Motifs- off-topic

Hi Yuri,

I don't know of a cheat-sheet, but I find the Introduction to Protein
Structure book by Branden and Tooze to useful for illustrations of
common folds.

Jeff

On Fri, Jan 20, 2012 at 10:13 AM, Yuri Pompeu yuri.pom...@ufl.edu wrote:
 Hello Everyone,
 Does anyone know of a quick (yet somewhat reliable) sort of cheat-sheet/quick 
 reference sheet with the more common folds with an illustrative example?

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Metal won't strip from IMAC

2012-01-12 Thread Michael Thompson
Katherine,

You are not alone. I have inadvertently destroyed a GE HisTrap column with high 
concentrations of proteins that contain many exposed cysteines. In my case the 
Co2+ resin turned a very dark purplish-brown and the protein appeared to have 
crashed out on the column. I didn't try to strip it, because I figured it was 
done for anyway, so I can't tell you any more about the problem. Here's how I 
explained it to myself (whether or not this is actually right I'm not 100% 
sure, but it makes sense in my head). The columns I was using have a maximum 
concentration of 5mM for DTT and 10mM for B-mercaptoethanol. So that seems like 
the column can handle 10mM thiol groups. If you have a protein with many 
cysteines and it is very highly concentrated (as was the case for me) then you 
are adding considerably more thiol groups to the solution. This abundance of 
thiols reduces the metal on the column, and disaster ensues. For me, repeating 
the same prep with less DTT (3mM vs. 5mM) in the buffer fixed the issue. If you 
are concerned about your protein oxidizing at lower concentrations of DTT or 
BME, the other alternative is to switch to TCEP. The IMAC columns can tolerate 
higher concentrations of TCEP, and it is a far superior reducing agent (more 
stable, more reductive, etc.)...but also a lot more expensive (although you can 
get away with using much less because it works so much better).

HTH,

Mike 


- Original Message -
From: Katherine Sippel katherine.sip...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, January 12, 2012 4:01:10 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Metal won't strip from IMAC

Hi all, 

I've run into a bit of a protein purification conundrum and wondered if anyone 
had encountered a similar situation. I've exercised all of my google-fu and 
can't find anything. It's a fairly straightforward setup; His-tagged protein 
and Talon Co2+ resin, load lysate, wash with 5 mM imidazole, elute with 150 mM 
imidazole. There is protein in the elution fractions as would be expected. The 
strangeness occurs when I try to regenerate the column. Using the standard 
protocol of 25 mM MES, 100 mM NaCl pH 5 doesn't change the color of the resin 
back to light pink the way it should with a regenerated column. I try stripping 
with the suggested 0.2M EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 
4% CHAPS and then EDTA, still pink, 1 M NaOH then EDTA, still pink. I've 
checked the resin using a Western (with a really specific monoclonal Ab) and it 
seems that my protein has somehow irreversibly bound to the column and is 
preventing the metal from releasing the sepharose. I've even tried competing 
the protein off with excess Co2+ and Mg2+ (the endogenous divalent bound 
cation). 

Clearly the solution is swapping to a Ni column, but this is really bugging me 
now. Has anyone run into this problem with IMAC before? 

Background: The protein does bind divalent cations (Mg and Mn) with low 
affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues 
total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers. 

Thanks, 

Katherine 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Pore Dimension Convention

2011-12-14 Thread Michael Thompson
Hi Jacob,

There are a number of programs that can calculate the radius of a pore. The one 
that comes to mind is called HOLE, and it can make a nice plot of the 
y-coordinate along the pore vs. pore radius. I don't recall exactly how this 
calculation is done, I think it is somehow related to the SASA (some sort of 
spherical probe type of thing), but I'm sure you can have a look at the 
documentation for more details. 

A quick search also shows that there are apparently a number of tools out there 
that will do similar analyses. See this web page for a summary:

http://www.caver.cz/index.php?sid=133

Good luck,

Mike




- Original Message -
From: Jacob Keller j-kell...@fsm.northwestern.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, December 14, 2011 3:14:33 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Pore Dimension Convention

Dear Crystallographers,

is there a convention for denoting/measuring pore sizes in protein
structures? Maybe inter-atom distances minus van der Waals radii?

JPK

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

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

2011-11-21 Thread Michael Thompson
- Forwarded Message -
From: Michael Thompson mi...@chem.ucla.edu
To: e dodson e.dod...@ysbl.york.ac.uk
Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

A question regarding the plea for less MR (which I support):

There have been several recent instances in which I have used the solution of 
an isomorphous structure to do rigid body refinement for a new crystal (as 
described by Eleanor). It has always produced good results. My question is 
about how to best handle the free set of reflections when doing this? I have 
heard a number of differing opinions about whether or not it is important to 
carry the freeR flags from the original structure over to the new data set. I 
have heard equally convincing arguments from both sides, so my young and 
impressionable mind does not know who to believe. I was hoping I could get an 
opinion from the advocates for less MR.

Sorry for hijacking this thread, but hopefully it will provide some insight 
that is relevant to the original post.

Thanks!

Mike




- Original Message -
From: Eleanor Dodson c...@ysbl.york.ac.uk
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

Just a plea for less molecular replacement.

If you get a new crystal of a known protein with the  same cell 
dimension as youur old crystal, the most likely scenario is that it has 
the same group, and you really should not try MR - use the previous 
solution as input to do rigid body refinement, and then
  a) the R factor will tell you if this is a reasonable hypothesis (it 
usually is..) and
b) you dont have this awful problem of not being able to compare the 
solutions..

  Eleanor

On 11/20/2011 03:57 PM, Napoleão Valadares wrote:
 Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
 Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.

 I think I understand it now, I always thought the one ring to rule them
 all translated in the crystallography realms to one origin to rule
 them all. That probably means I have a long road in front of me.

 I'm still half confused, I definitely need to read more, as much as I
 read about symmetry and space groups I never seem to improve or get a
 better understanding, but I'll keep trying.

 About the same origin:
 The pdbs of both Solution-1 and Solution-2 present the same space group
 and cell, as observed opening the pdbs as text files or in pymol. When I
 open both maps on coot they are not superposed but present the same cell
 and origin.

 If I open both solutions on pymol they clash. If I generate the symmetry
 mates of both solutions none of them are superposed, instead they clash.
 But I think they are related as you all pointed, I'll check it out.

 Thank you all for your kind answers and your patience with a beginner.
 Regards from a sunny Brazil,
 Napo


 On 11/20/2011 2:58 AM, Felix Frolow wrote:
 Napoleao,
 It is so called alternative origins play a game with you. You do not
 change your structure by shifting 1/2 translation (or even combination
 of these translations)
 into directions of the main axes of your unit cell. Structure factors
 after this operation stay the same, however phases change
 systematically, producing however the same
 map features.
 Would I be a begin crystallographer now, I would read a bit more old
 fashioned books
 on crystallography such as probably Jensen and Stout…
 FF
 Dr Felix Frolow
 Professor of Structural Biology and Biotechnology
 Department of Molecular Microbiology
 and Biotechnology
 Tel Aviv University 69978, Israel

 Acta Crystallographica F, co-editor

 e-mail: mbfro...@post.tau.ac.il mailto:mbfro...@post.tau.ac.il
 Tel: ++972-3640-8723
 Fax: ++972-3640-9407
 Cellular: 0547 459 608

 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:

 Hello,
 I'm observing a very strange phenomena (at least to me, I'm a
 beginner). It is related to symmetry (I think).

 I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas  35% in
 the last shell) and a partially refined solution with R/Rfree 22/24,
 166 aminoacids observed and around 30 solvent molecules. I'll call
 this Solution-1. The refinement was smooth, the densities were very
 clearly asking for the correct missing side chains and the map
 looks good.

 The space group I'm using is P212121, pointless and XDS agree with
 that (but me and pointless both have a long history of being wrong
 about space groups). Phenix.xtriage says there's no twinning.

 I took Solution-1 and used it as a template in a molecular
 replacement in the same space group (P212121) using the same mtz as
 the one used to refine the template. I got a different (not
 superposed in space) solution (called Solution-2, scores by Phaser
 RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined
 in Phenix to R/Rfree 24/26 without any solvent molecule

Re: [ccp4bb] Protein preps become a jelly

2011-08-30 Thread Michael Thompson
To add to what Pat has mentioned, I work with a protein that has a number of 
exposed Cys residues, and it turns into a gel at 4 degrees within a week, even 
if I store it in buffer with reducing agents. This happens every time I store 
it at 4 degrees. To test if your jelly is due to S-S crosslinking, add a 
really huge excess of DTT or beta-mercaptoethanol to a small sample and see if 
it goes back into solution. I usually add 100mM DTT for this test. If it does 
turn out to be disulfide-related, you can try to add reducing agents with 
longer half-lives (ie TCEP instead of DTT), but unfortunately you may just have 
to work with the protein within a couple days of finishing the prep.

Mike




- Original Message -
From: Patrick Loll pat.l...@drexel.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, August 30, 2011 9:06:52 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Protein preps become a jelly

Certainly not unprecedented, or even that unusual (I remember making gels from 
BSA and IgG solutions during grad school rotations).  Gel formation usually 
requires crosslinking, so consider whether you might be getting adventitious 
disulfide bond formation.
Pat

On 30 Aug 2011, at 11:31 AM, aidong wrote:

 Dear Buddies,
 
 Sorry for bothering you with an off-ccp4 question.  We recently are 
 experiencing a very strange phenomena.  A couple of protein preps with 
 reasonably high concentration (10-20mg/ml) become a jelly after storages for 
 overnight or a couple of days at 4C.  All of them have been purified by gel 
 filtration.  Some of these proteins behave like this from very first preps 
 but some of them had been very kind to us previously.  We have googled 
 extensively in CCP4BB and www but it appears this only happens to us.  It 
 would be highly appreciated that you could exchange their experiences or 
 provide your suggestions.
 
 Aidong Han, Ph.D
 
 Department of Biomedical Sciences
 School of Life Sciences
 Xiamen University
 Xiamen, Fujian 361005
 China
 Phone: 0592-218-8172 (O)
  0592-218-8173 (L)
 Web: http://life.xmu.edu.cn/adhanlab/

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Detergents to eliminate non specific aggregations

2011-08-27 Thread Michael Thompson
Hampton makes a Detergent Screen that works the same way as the Additive 
Screen. 

http://hamptonresearch.com/product_detail.aspx?cid=1sid=39pid=31

It has 96 unique detergent reagents for crystal screening. 

It is a bit expensive, but if you don't want to purchase the whole kit you 
could just download the formulation table and then you will have a nice long 
list of possibilities.

Mike


- Original Message -
From: 商元 shangyuan5...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Saturday, August 27, 2011 4:55:27 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Detergents to eliminate non specific aggregations

Hi, everyone, 
I can get my protein complex but there are some non-specific aggregation from 
the NMR spectra, and chaps can improve it. 
So, besides chaps, is there any other detergents to be used during crystal 
screening? All suggestions are welcome. 

ThanksRegards, 
Yuan 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Off topic_protein degradation.

2011-08-19 Thread Michael Thompson
Another option is DTNB (aka Ellman's Reagent). This compound reacts with free 
thiols and produces a yellow color. The reaction is stoichiometric, so if you 
have access to a very basic spectrophotometer and know your protein 
concentration you can quantify the free thiols quite easily.

Basic pH doesn't break disulfides, if anything it stabilizes them. I would be 
more concerned that they are incorrectly formed as described by Herman.

Mike



- Original Message -
From: D Bonsor dbon...@ihv.umaryland.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, August 19, 2011 6:21:41 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Off topic_protein degradation.

If you are unsure about whether the disulfides have formed treat a small amount 
of protein with N-ethylmaleimide. If the disulfides have not formed, when you 
perform mass spec on the protein you should see an increase of mass of 125Da 
for each exposed cysteine.

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] anomalous scatterer

2011-08-11 Thread Michael Thompson
Hello,

Regarding your empty Tb site: How similar are the two copies of the protein in 
the ASU? You can overlay the two individual copies from the ASU to check and 
see if the binding site without density has been distorted somehow so that it 
can no longer accomodate the Tb, maybe due to crystal packing, etc.

Mike




- Original Message -
From: Huiming Li huimin...@hotmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, August 9, 2011 9:38:39 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] anomalous scatterer


Hi All, 

I am working on a Tb binding protein on which I collected anomalous data at Tb 
edge of 1.648 A. Each protein is designed to bind one Tb. There are two copies 
of the protein in an ASU. I have two questions. First, I am only able to see 
one copy of the protein with Tb bound, and no density on the other copy. Isn't 
this a bit surprising? Second, there is one additional peak on each monomer at 
the site where Fe is known to bind, and Fe has an edge of 1.739A. At 1.648A, f' 
and f'' of Fe is only about 1/5 of Tb. Is it possible Fe also shows some 
anomalous signal at Tb edge? 

Thank you, 

Huiming Li, Ph.D. 
Immune Disease Institute 
Children's Hospital Boston 
Harvard Medical School 
Boston, MA 02115 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] Intensity-Weighted Reciprocal Lattice

2011-07-21 Thread Michael Thompson
Hello ccp4  phenix BB members,

I would like to view the intensity-weighted reciprocal lattice for several data 
sets that I have collected. (The data have been indexed, integrated and scaled 
with Denzo and Scalepack.) I was wondering if anyone could offer some advice on 
what might be the best and/or most practical way to do this?

I know that there are several programs out there that can generate sections 
(i.e. 0,k,l) of the reciprocal lattice, such as LABELIT and xrayplot. Are there 
any other options for doing this, perhaps within ccp4 and/or phenix? I once saw 
someone give a presentation and they had a little video that showed a three 
dimensional section of the reciprocal lattice rocking back and forth, which was 
really cool. I liked this because I felt like it gave a much more holistic 
representation as opposed to viewing a bunch of individual sections. I don't 
know if there is an easy way to do this, or if this person somehow managed to 
create this 3D depiction from a series of sections.

Any tips or recommendations would be appreciated.

Thanks,

Mike




-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] peptide docking

2011-07-12 Thread Michael Thompson
I have attempted to use the RosettaDock webserver for protein-protein docking. 
If I recall correctly, the webserver is only capable of doing fixed backbone 
docking simulations. If you are docking a small peptide with a protein I 
suspect you want the peptide to be somewhat flexible. Although my results from 
the RosettaDock webserver were disappointing, the more recently developed 
PyRosetta (pyrosetta.org) is extremely powerful and is certainly capable of 
doing what you need. PyRosetta is a Python interface to Rosetta, so it requires 
some knowledge of Python and you need to have the software installed locally, 
but you can get it to do lots and lots of useful things by writing relatively 
short scripts. I have had poor luck using webservers for docking, because I 
have found that often you need to tweak parameters (weights, restraints, etc.) 
here and there to get the simulations to give sensible results for a particular 
set of partners and webservers do not give you enough control of the variables.

Good luck,

Mike



- Original Message -
From: Juergen Bosch jubo...@jhsph.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 11, 2011 6:14:20 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] peptide docking

Here are some options: 
http://vina.scripps.edu/ 
http://rosettadock.graylab.jhu.edu/ 


Jürgen 



On Jul 11, 2011, at 8:24 PM, crystal wrote: 


Hi Pius, 
Thanks for the tip. I've never used that utility in PyMol. It'll give it go! 
From the drop-down menu of the Builder tab, it seems like one could specify 
the secondary structure as well. 
Have you ever tried that option? 
Thanks. 


On Mon, Jul 11, 2011 at 12:43 PM, Pius Padayatti  ppadaya...@gmail.com  
wrote: 


I am just guessing this what you want. 
a peptide of certain length say 10 aa and need it to be built. 
use pymol Builder utility and choose protein and build your peptide. 
you do not need any density. 
write out the co-ordiantes and use it in autodock for docking. 
pius 





On Mon, Jul 11, 2011 at 12:41 PM, crystal  aac...@gmail.com  wrote: 
 Hi all, 
 Thanks for all the helpful suggestions. It seems like Phenix LigandFit 
 requires density for the ligand but unfortunately I only have the 
 ligand-free model. I've tried blasting the peptide sequence using the 
 sequence search tool in the PDB but failed to get any hits. I was 
 wondering how one could grep it out of a set of PDB files like Robbie has 
 mentioned. Since there is relatively high conformational flexibility, I was 
 thinking about starting with a 5-mer. Does that sound reasonable with people 
 who have experience with peptide docking? Pius mentioned about making the 
 model in Coot, PyMol etc. Do you need density in order to fit the ligand or 
 can one just mutate residues along a strand in an existing model to the 
 desired sequence and use that fragment for docking? 
 Thanks for the helpful feedback, everyone. 
 
 
 On Sun, Jul 10, 2011 at 8:54 PM, Joel Tyndall  joel.tynd...@otago.ac.nz  
 wrote: 
 
 How big is the peptide? 
 
 
 
 _ 
 
 Joel Tyndall, PhD 
 
 Senior Lecturer in Medicinal Chemistry 
 National School of Pharmacy 
 University of Otago 
 PO Box 56 Dunedin 9054 
 New Zealand 
 
 Skype: jtyndall 
 http://www.researcherid.com/rid/C-2803-2008 
 
 Pukeka Matua 
 Te Kura Taiwhanga Putaiao 
 Te Whare Wananga o Otago 
 Pouaka Poutapeta 56 Otepoti 9054 
 Aotearoa 
 
 Ph / Waea +64 3 4797293 
 Fax / Waeawhakaahua +64 3 4797034 
 
 
 
 From: CCP4 bulletin board [mailto: CCP4BB@JISCMAIL.AC.UK ] On Behalf Of 
 crystal 
 Sent: Saturday, 9 July 2011 1:09 p.m. 
 
 To: CCP4BB@JISCMAIL.AC.UK 
 Subject: [ccp4bb] peptide docking 
 
 
 
 Hi all, 
 As I'm a newbie in peptide docking, I was wondering what 
 programs/servers people would suggest for generating the peptide PDB 
 (keeping all the proper stereochemistry)? 
 Is it possible to extract a file from the PDB database? 
 All comments will be much appreciated! 
 






-- 
Pius S Padayatti,PhD, 
Phone: 216-658-4528 







.. 
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/ 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] The Good and the Bad crystal contact?

2011-06-29 Thread Michael Thompson
Hi Paul,

While crystal contacts are typically of a non-covalent nature, there are some 
exceptions. A disulfide bond can act as a crystal contact, which is a covalent 
interaction. A technique called synthetic symmetrization involves the 
engineering of single cysteine mutants, followed by oxidation to form an 
intermolecular disulfide bond that lies on a 2-fold symmetry axis between the 
two monomers. The original goal of this technique was to turn an asymmetric 
monomer into a symmetric dimer, which should crystallize more readily if the 
artificial 2-fold lies on a crystallographic symmetry axis. Recently, a paper 
illustrated that even if the artificial 2-fold axis does not become a 
crystallographic axis, the introduction of a disulfide can create artificial 
crystal contacts, which also aid in crystallization. Check out the following 
paper. There is a nice figure that shows two protein molecules that are really 
not touching with the exception of the engineered disulfide bond that forms the 
only contact.

Protein Sci. 2011 Jan;20(1):168-78.
Synthetic symmetrization in the crystallization and structure determination of 
CelA from Thermotoga maritima. 

HTH,

Mike




- Original Message -
From: Paul Lindblom lindblom.p...@googlemail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, June 29, 2011 2:22:26 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] The Good and the Bad crystal contact?

Hi everybody, 

can anybody tell me how crystal contacts are defined? Are there good and bad 
crystal contacts? They are the most important interactions with impact on the 
crystal quality, but they are not of covalent nature, aren´t they? 

With best regards, 

Paul 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Crystal structure and NMR structure

2011-05-24 Thread Michael Thompson
It's not a protein, but here's another example: the PreQ1 Riboswitch. In this 
case the two methods revealed different structures, but the NMR group was able 
to determine that the differences were due to calcium in the crystallization 
condition.

Crystal Structure:

Nat Struct Mol Biol. 2009 Mar;16(3):343-4. Epub 2009 Feb 22.
Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot 
recognizing an essential hypermodified nucleobase.
Klein DJ, Edwards TE, Ferré-D'Amaré AR.

NMR Structure:

Mol Cell. 2009 Mar 27;33(6):784-90. Epub 2009 Mar 12.
Structural Insights into riboswitch control of the biosynthesis of queuosine, a 
modified nucleotide found in the anticodon of tRNA.
Kang M, Peterson R, Feigon J.
Erratum in Mol Cell. 2010 Aug 27;39(4):653-5. 

An explanation of the differences:

J Am Chem Soc. 2011 Apr 13;133(14):5190-3. Epub 2011 Mar 16.
Comparison of solution and crystal structures of preQ1 riboswitch reveals 
calcium-induced changes in conformation and dynamics.
Zhang Q, Kang M, Peterson RD, Feigon J.


Cheers,

Mike



- Original Message -
From: Vandu Murugan wandumuru...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, May 20, 2011 11:34:31 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Crystal structure and NMR structure

Dear all, 
I would like to get some information on proteins where there is 
conformation/structural change between the crystal structure and solution 
structure of the same protein. Do anybody came across such situations? Thanks 
in advance.. 

cheers, 
Vandu 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Dehydration treatments

2011-05-03 Thread Michael Thompson
Israel,

Here is a paper that describes a phenomenon like what you have observed:

Structure. 2003 Feb;11(2):139-45.
Dehydration converts DsbG crystal diffraction from low to high resolution.
Heras B, Edeling MA, Byriel KA, Jones A, Raina S, Martin JL.


And another review that touches briefly on the idea, among other things:

Acta Crystallogr D Biol Crystallogr. 2005 Sep;61(Pt 9):1173-80. Epub 2005 Aug 
16.
Post-crystallization treatments for improving diffraction quality of protein 
crystals.
Heras B, Martin JL.

Mike


- Original Message -
From: Israel Sanchez israelsan...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Sunday, May 1, 2011 10:32:21 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Dehydration treatments

Hi folks, 


I am currently impressed by the efficiency of dehydration treatments over the 
diffraction capacity of our crystals in one particular condition. Without any 
treatment the crystals seldom diffract to 20-30A but in our last synchrotron 
trip the very same crystals, after been incubated with increasing concentration 
of low molecular weight PEGs diffracted to 6A. 

I was wondering if anyone has studied these effects in a systematic way. Does 
anyone on the ccp4bb knows references or has any experience/pseudo-religious 
believes that do not care to share with the community about this particular 
topic? 


Thank you very much in advance 


-- 
Israel Sanchez Fernandez PhD 
Ramakrishnan-lab 
MRC Laboratory of Molecular Biology, 
Hills Road, Cambridge, CB2 0QH, UK 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] problem with pdb its symmetry molecule

2011-04-26 Thread Michael Thompson
Ramanuj,

This is definitely possible. Cases of intertwined homodimers are rare, but 
there are several known structures that demonstrate this phenomenon, and they 
are very interesting (especially with respect to studying knotted proteins - 
see reference below). Examples are pdb IDs: 2ouf, 1myk, 2rh3.

A cool paper regarding intertwined homodimers and knotted proteins:

Proc Natl Acad Sci U S A. 2010 Nov 30;107(48):20732-7. Epub 2010 Nov 10.
Structure and folding of a designed knotted protein.
King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO.

Your structure is very interesting.

Mike



- Original Message -
From: Ramanuj Banerjee ramanuj.baner...@saha.ac.in
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 26, 2011 12:37:29 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] problem with pdb  its symmetry molecule


Dear All, I have solved a protein structure (experimentally phased) with 1 
molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
factor is .22 and R free .27 with Ramachandran favoured 98% and R and R free 
are decreasing with refinement.The problem is: when the pdb is opened in pymol 
and symmetry mates generated, the upper part of the molecule shows to be 
intertwined with the symmetry molecule (attached .jpg), but there are no 
clashes in between the two.The electron density is so very fine that no 
alternative choices of chain flow are available.All the processes starting from 
phasing and refinement have been done in Phenix.Is such a thing possible ? 

[image/jpeg:a copy.jpg]

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] problem with pdb its symmetry molecule

2011-04-26 Thread Michael Thompson
Ramanuj,

This is definitely possible. Cases of intertwined homodimers are rare, but 
there are several known structures that demonstrate this phenomenon, and they 
are very interesting (especially with respect to studying knotted proteins - 
see reference below). Examples are pdb IDs: 2ouf, 1myk, 2rh3.

A cool paper regarding intertwined homodimers and knotted proteins:

Proc Natl Acad Sci U S A. 2010 Nov 30;107(48):20732-7. Epub 2010 Nov 10.
Structure and folding of a designed knotted protein.
King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO.

Your structure is very interesting.

Mike


- Original Message -
From: Ramanuj Banerjee ramanuj.baner...@saha.ac.in
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 26, 2011 12:37:29 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] problem with pdb  its symmetry molecule


Dear All, I have solved a protein structure (experimentally phased) with 1 
molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
factor is .22 and R free .27 with Ramachandran favoured 98% and R and R free 
are decreasing with refinement.The problem is: when the pdb is opened in pymol 
and symmetry mates generated, the upper part of the molecule shows to be 
intertwined with the symmetry molecule (attached .jpg), but there are no 
clashes in between the two.The electron density is so very fine that no 
alternative choices of chain flow are available.All the processes starting from 
phasing and refinement have been done in Phenix.Is such a thing possible ? 

[image/jpeg:a copy.jpg]

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] OT: Covalent modification of Cys by reducing agents?

2011-04-15 Thread Michael Thompson
Hi All,

I was wondering if anyone knew whether or not it is possible for reducing 
agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form 
covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have 
some puzzling biochemical results, and in the absence of a structure (thus 
far), I was wondering if this might be something to try to control for. I have 
never heard of this happening (or seen a structure where there was density for 
this type of adduct), but I can't really think of a good reason for why this 
wouldn't happen. Especially for something like BME, where the molecule is very 
much like the Cys sidechain and seems to me like it should have similar 
reactivity. The only thing I can think of is if there is a kinetic effect 
taking place. Perhaps the rate of diffusion of these small molecules is much 
faster that the formation of the S-S bond?

Does anyone know whether or not this is possible, and why it does or does not 
happen?

Thanks,

Mike




-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] methods to capture proteins from cell culture medium

2011-04-12 Thread Michael Thompson
Bei,

I had a former labmate who had the same situation and would load somewhere 
between 6-8L of media directly onto a column. I don't remember what type of 
column it was, ion exchange may not be ideal if the ionic strength of your 
medium is high. I think it may have been a phenyl sepharose column.

Good luck,

Mike



- Original Message -
From: joybeiyang joybeiy...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] methods to capture proteins from cell culture medium



Dear all, 

My protein of interest was expressed as secreted protein, so I have to collect 
the medium and change the buffer with sortorius Jet before I load the sample 
onto a IMAC, the buffer change step in my current protocol can last for 12hrs 
(I have to concentrate 4L to 200ml, then dilute it with lysis buffer and 
concentrate it again, then dilute and concentrate repeatedly) and is really 
boring and troublesome, besides I always observe protein loss during this step 
and the detergent in the medium usually concentrate as well in this step which 
would interfere with subsequent purification process. I am wondering if there 
are more convenient ways to capture the target protein from medium? How about 
the following: 

1. directly load the medium onto a ion exchange column? 

2. Amonium sulfate precipitation? 

3. anyother thoughts? 

Thank you very much in advance! 

Best, 

Bei 
2011-04-12 

joybeiyang 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue

2011-04-09 Thread Michael Thompson
It is not surprising that your bradford and BCA assays don't agree if you have 
no aromatic amino acids in your protein. Bradford dye binds to hydrophobic 
residues, mainly aromatics, so I would guess your bradford is consistantly 
giving lower measurements than the BCA assay. I also wouldn't be surprised if 
the results of your Bradford vary significantly between replicates. The BCA 
assay reagent interacts with the backbone amides, not with any sidechains, so I 
would tend to believe that measurement more than anything else you have done. 

I work with a protein that has very few hydrophobics (only one aromatic - a 
Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to 
be consistent.

Mike




- Original Message -
From: Arpit Mishra ar...@igib.in
To: CCP4BB@JISCMAIL.AC.UK
Sent: Saturday, April 9, 2011 2:52:21 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue

hello everybody 


i am working on the protien which dont have any aromatic residue i do fplc 
other purification using 220 absorption, but i want to quantitate protein 
precisely i have tried using BCA nd bradford but both methods quantification is 
not matching,,so any one is having sum idea how to quantitate it precisely 


thanks in advance for your valuable suggestion.. 




Arpit Mishra 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue

2011-04-09 Thread Michael Thompson
John,

I believe my statement is accurate. The Compton  Jones paper you cite states 
in the abstract: Interactions are chiefly with arginine rather than primary 
amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and 
Phe) give slight responses. The binding behavior is attributed to Van der Waals 
forces and hydrophobic interactions. Also, 23 years later, the following paper 
provides a detailed study of the mechanism of Coomassie Brilliant Blue G-250 
binding to proteins and discusses the hydrophobic interactions I mentioned 
between aromatic residues and the dye.

Georgiou, et al. Anal Bioanal Chem. 2008 May;391(1):391-403. 

Cheers,

Mike



- Original Message -
From: John A. Newitt newit...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Saturday, April 9, 2011 12:06:23 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] how to quantitate protein which dont have ne aromatic 
residue

At 9:47 AM -0700 4/9/11, Michael Thompson wrote:

Bradford dye binds to hydrophobic residues, mainly aromatics,

The statement above is not accurate.

Compton and Jones. Anal. Biochem. 151(2): 369-374, 1985

- John
-- 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

2011-04-05 Thread Michael Thompson
dengzq1987,

I am also working on a data set that has this exact alignment/mosaicity problem 
noted by others. If you make a movie of your dataset that displays your images 
in series, or if you just click through them quickly in adxv or your program of 
choice, you will see that very few of the spots ever leave their positions on 
the detector as you rotate the crystal. This demonstrates the mosaicity issue 
described by Bert and Ed. In my case, Denzo seems to be able to properly 
measure the a and b axes of my hexagonal cell (I know this based on a 
comparison of these values to structures of homologs in the same SG), but tells 
me that my c axis is only 0.21A. It seems to me that in the case of overlaps, 
the dimensions of the corresponding axis would be overestimated rather than 
seriously underestimated (0.21A!), so I'm not really sure of what is going on 
here. Maybe someone who knows more about the software could shed some light on 
this crazy measurement? I've been told by a few people that XDS may be able to 
handle this dataset, but I haven't gotten to trying it yet. I'll work on it 
today or tomorrow and follow up if I have any success. 

Best,

Mike






- Original Message -
From: Edward A. Berry ber...@upstate.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 5, 2011 8:38:17 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

In the first image (spot 2) the long unit cell is obvious from the closely 
spaced
spots, but this kind of image can be handled easily by Denzo. The second one
(spot 1), although at first glance the spots seem well separated, is the 
problem.
As Bert pointed out the mosaicity is high, so the lunes are overlapping
(you don't see separate rings or crescents where Ewalds sphere intersects
different layers of the lattice). That means the apparently individual
spots are multiple spots one on top of the other, with the long axis
nearly perpendicular to the plane of the picture. This means there is
no information about the spacing of the long axis in this picture,and
the picture is rather insensitive to rotation, so integration is
likely to get lost unless you fix these parameters. If you fix the
parameters based on the good orientation and integrate on through,
you will be sampling the lattice rods at intervals (as the 2D electron
crystallographers say) rather than measuring discreet spots.
However if you collect while rotating around the long axis, you will
never see this orientation and should have no serious problem.
eab

dengzq1987 wrote:
 hello Jürgen_Bosch,
 because i have collected the data,but can't index.in some direction,the
 spot is separated .but the others are set close together(picture spot1
 and spot 2).so we think there is one long unit cell axes.
 2011-04-05
 
 dengzq1987
 
 *发件人:* Jürgen_Bosch
 *发送时间:* 2011-04-05 20:29:18
 *收件人:* dengzq1987
 *抄送:* CCP4BB@JISCMAIL.AC.UK
 *主题:* Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?
 What do you consider long ? 200, 300 ? 600 A ? Before shooting try to
 run strategy or xplan. Move the detector back to first reliably be able
 to determine your cell. Then double your estimated mosaicity and see
 what strategy suggests. If you don't get many overlaps (5%) then try a
 closer distance. Don't rotate 1degrees but take 1/2 of the mosaicity.
 Obviously you want to make good use of the detector area so adjust the
 edges to where your crystal really diffracts. And if that resolution
 leads to too many overlaps then limit your resolution and get first a
 good datasets home. You then can play with 2theta for a higher
 resolution dataset.
 Another obvious thing to do and you don't mention what reduction program
 you use is to let XDS sort your problem out. Unless you collected to
 high resolution without being cautious XDS could help. If not, well then
 you had your experience and now should know better.
 SSRL has options to collect 450 A cells to 3A without much hassle. That
 was my largest cell so far.
 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 Apr 5, 2011, at 1:05, dengzq1987 dengzq1...@gmail.com
 mailto:dengzq1...@gmail.com wrote:

 hello all,
 does anyone have the experience of Collecting Data from Long Unit Cell
 Axes ? I have a crystal that diffracts to about 4 A. in some
 /direction/ the spots overlap. we can't use the data to index .we
 think it is because that there is a long unit cell axes. so is there
 any method to solve this problem?
 best wishes.
 2011-04-05
 

Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

2011-04-05 Thread Michael Thompson
Thanks for the explanation Ed, that makes sense.


- Original Message -
From: Edward A. Berry ber...@upstate.edu
To: mi...@chem.ucla.edu
Cc: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 5, 2011 12:22:21 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

When the c axis is perpendicular to the image, i.e. parallel to the xray beam,
the only information about spacing along the c axis comes from the distance
between the lunes. There should be a circle of spots around the direct beam
with l=0, the a clear space, then a ring with l=1, then a clear space, then
a ring with l=2 and so on. As the c dimension gets longer, the layers get
closer together in reciprocal space, and these rings caused by intersection
of the l-planes with Ewald's sphere get closer together.
As the mosaicity increases, reflections whose centers are farther from ewalds
sphere will be in diffraction, and the rings get broader. In the extreme
of long c axis and/or high mosaicity the rings overlap completely and there
appears to be only one circle of spots around the direct beam center extending
out to the diffraction limit. Presumably denzo takes this to be the l=0 layer,
and since it can't find the l=1 layer assumes it is out of the picture beyond
the diffraction limit, which would be the case if you had an extremely
short c axis so the layers are far separated.
So it can get a good fit to the spot using correct a, b and a very small c.

eab

mi...@chem.ucla.edu wrote:
 dengzq1987,

 I am also working on a data set that has this exact alignment/mosaicity 
 problem noted by others. If you make a movie of your dataset that displays 
 your images in series, or if you just click through them quickly in adxv or 
 your program of choice, you will see that very few of the spots ever leave 
 their positions on the detector as you rotate the crystal. This demonstrates 
 the mosaicity issue described by Bert and Ed. In my case, Denzo seems to be 
 able to properly measure the a and b axes of my hexagonal cell (I know this 
 based on a comparison of these values to structures of homologs in the same 
 SG), but tells me that my c axis is only 0.21A. It seems to me that in the 
 case of overlaps, the dimensions of the corresponding axis would be 
 overestimated rather than seriously underestimated (0.21A!), so I'm not 
 really sure of what is going on here. Maybe someone who knows more about the 
 software could shed some light on this crazy measurement? I've been told by a 
 few people that
 
 XDS may be able to handle this dataset, but I haven't gotten to trying it yet. 
I'll work on it today or tomorrow and follow up if I have any success.

 Best,

 Mike






 - Original Message -
 From: Edward A. Berryber...@upstate.edu
 To: CCP4BB@JISCMAIL.AC.UK
 Sent: Tuesday, April 5, 2011 8:38:17 AM GMT -08:00 US/Canada Pacific
 Subject: Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

 In the first image (spot 2) the long unit cell is obvious from the closely 
 spaced
 spots, but this kind of image can be handled easily by Denzo. The second one
 (spot 1), although at first glance the spots seem well separated, is the 
 problem.
 As Bert pointed out the mosaicity is high, so the lunes are overlapping
 (you don't see separate rings or crescents where Ewalds sphere intersects
 different layers of the lattice). That means the apparently individual
 spots are multiple spots one on top of the other, with the long axis
 nearly perpendicular to the plane of the picture. This means there is
 no information about the spacing of the long axis in this picture,and
 the picture is rather insensitive to rotation, so integration is
 likely to get lost unless you fix these parameters. If you fix the
 parameters based on the good orientation and integrate on through,
 you will be sampling the lattice rods at intervals (as the 2D electron
 crystallographers say) rather than measuring discreet spots.
 However if you collect while rotating around the long axis, you will
 never see this orientation and should have no serious problem.
 eab

 dengzq1987 wrote:
 hello Jürgen_Bosch,
 because i have collected the data,but can't index.in some direction,the
 spot is separated .but the others are set close together(picture spot1
 and spot 2).so we think there is one long unit cell axes.
 2011-04-05
 
 dengzq1987
 
 *发件人:* Jürgen_Bosch
 *发送时间:* 2011-04-05 20:29:18
 *收件人:* dengzq1987
 *抄送:* CCP4BB@JISCMAIL.AC.UK
 *主题:* Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?
 What do you consider long ? 200, 300 ? 600 A ? Before shooting try to
 run strategy or xplan. Move the detector back to first reliably be able
 to determine your cell. Then double your estimated mosaicity and see
 what strategy suggests. If you don't get many overlaps (5%) then try a
 closer 

Re: [ccp4bb] Data Strategy for anisotropic diffraction

2011-04-05 Thread Michael Thompson
Alex,

You should check out this webserver: http://services.mbi.ucla.edu/anisoscale/

If it cannot solve your problem, it will at least point you in the direction of 
some answers.

Good luck,

Mike


- Original Message -
From: Alex Barth alexbart...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 5, 2011 4:11:18 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Data Strategy for anisotropic diffraction

Dear all, 

i am new in the field and my crystals give anisotropic pattern. 

I would really appreciate if someone who has experience with data collection 
with anisotropy diffraction to recommend relative literature on data strategy 
concerning anisotropy. 

Cheers, 
Alex 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] Synthetic RNA for Crystallization

2011-03-13 Thread Michael Thompson
Hello All,

I am looking for some advice from some experienced RNA crystallographers. I 
would like to order some relatively short (90 bases) synthetic RNAs for 
crystallization trials. I was wondering if anyone could comment on the use of 
synthetic RNAs for crystallization. Specifically, what is the longest synthetic 
RNA that can be used for crystallization trials? I've seen some structures in 
the PDB that are up to 88 bases and are reported to have been obtained with 
synthetic constructs (3OWI - glycine riboswitch), but I don't really know if 
that's routine or if it's an exceptional case. Also, for those who have 
experience with the use of synthetic RNAs, I was wondering where people 
generally order their synthetic constructs from? Our resident expert in RNA 
crystallography recommended a company called Dharmacon (part of ThermoFisher), 
but I was hoping that I might get some other opinions as to which companies 
make the best quality oligonucleotides, provide samples with the highest 
purity, and have the most reasonable prices.

Thanks in advance for the help!

Mike



-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] off-topic: tag removal

2011-02-24 Thread Michael Thompson
Hi Phil,

Depending on the characteristics of the c-terminal region of interest, you 
might try a carboxypeptidase. These enzymes cleave residues from the c-terminus 
and stop at various motifs, depending on the specific enzyme. There are several 
available commercially, each having a slightly different activity. Also, the 
following paper may be of interest:

Current strategies for the use of affinity tags and tag removal for the 
purification of recombinant proteins
José Arnau, Conni Lauritzena, Gitte E. Petersena and John Pedersena
Protein Expression and Purification, Volume 48, Issue 1, July 2006, Pages 1-13 

Good Luck,

Mike




- Original Message -
From: Philipp Ellinger filu...@gmx.de
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, February 23, 2011 1:17:33 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] off-topic: tag removal



Dear all, 


I have a question concerning removal of a his-tag sequence. 
We have crystallized a protein with an important feature at the C-terminal part 
of the protein. 
Unfortunately, we cannot express it with a N-terminal his-tag, only with a 
C-terminal his-tag. 


Therefore we are looking for a protease which cleaves off the sequence without 
leaving any extra amino acid on the C-terminus of our protein. Meaning we 
obtain really the wild type protein. 
Does anyone know about a protease or cleavage site which is completely removed? 




Many thanks in advance 


Phil 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] strange density

2011-02-24 Thread Michael Thompson
Jacob,

Roger is correct, this concept does refer to the Pearson HSAB theory. To 
summarize: This theory is applicable outside of inorganic chemistry as well, 
but it is extremely useful for explaining coordination chemistry of 
metal-ligand complexes. The theory states that hard acids interact with 
hard bases and soft acids interact with soft bases to form a bond that is 
covalent-like in nature. Hardness vs. softness is based on the energetic 
properties of the HOMO and LUMO of the acid and base. Generally hard 
acids/bases have small atomic/ionic radii, low polarizability, and high 
electronegativity whereas soft acids/bases tend to have larger radii, high 
polarizability, and low electronegativity. 

Hard bases are things like carboxylates, whereas soft bases are things like 
thiolates. Ligands with nitrogen (imidazole) are often in the middle somewhere.

Hard acids are ions like Na+, K+, Mg2+, etc., while soft acids are metals like 
mercury, silver, etc. Again, many biologically relevant things lie in the 
middle of the spectrum somewhere (Fe, Co, Zn).

It is possible to calculate the chemical hardness of a species, but that's 
where my knowledge stops.

Hope this is helpful,

Mike




- Original Message -
From: Jacob Keller j-kell...@fsm.northwestern.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, February 24, 2011 10:39:09 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] strange density

I have heard hard and soft many times now about O's and N's--to
what property of those ligands does this metaphor refer?

JPK

On Thu, Feb 24, 2011 at 12:47 PM, Jeffrey D Brodin jbro...@ucsd.edu wrote:
 Alex,

 I modeled in the bis-tris with the tertiary amine and and his imidazole
 coordinating axially and the four oxygens coordinating in the equatorial
 plane. However, it's hard for me to tell from your images if there are two
 His coordinating? Either way, that crescent shape could easily be explained
 by a bis-tris molecule, you'll just have to figure out how exactly to model
 it in. It's also possible that the metal is a Mg, but as people have already
 mentioned, nitrogens probably wouldn't coordinate very tightly to a hard
 metal. Lastly, I'm also not sure off the top of my head how tightly bis-tris
 binds metals, but it should be an easy number to look up. Hope this helps,

 Jeff
 On Feb 24, 2011, at 9:02 AM, Alex Singer wrote:

 Hi -- thank you for all your help.  The majority opinion seems to be a
 metal for the sphere (Ni from the Ni-affinity column, which (Joe
 Patel, correct) was used during purification, but Zn and Fe were also
 mentioned), and either water molecules, bis-tris or some other small
 molecule forming the crescent.  Just looking at the density, the
 occupancy would seem to be quite high, so I'm surprised that a Ni ion
 (or a contaminating metal ion) could have gone through the
 purification and still remained at high enough concentration to be
 clearly visible in the crystals.  However, I'll still try this but
 first some points of clarification and questions which you can either
 email me seperately or post to the the group.

 a.  it was collected at beamline 19-BM at Argonne, so radiation damage
 is an issue.  Thierry Fishmann -- for the gln residue, there was
 difference density for the gamma carbon after the first conformation
 was modeled in, thus the addition of the second conformation, which I
 agree is suspect.  What does the radiation damage do chemically and
 would that make the gamma carbon more mobile?

 b.  Jeffrey D Brodin -- how did you model in the bis-tris?  Looking at
 the bis-tris molecule from Hic-up, was the N at the centre of the
 crescent and the O6 and O8 at the edges?

 c.  JR Helliwell -- there are 4 molecules in the AU, but two H138's
 are pointing into the solvent.  Thus the molar ratio of protein
 molecules to thing 1 is 4:1.  Also looking at the images, I see no
 ice rings -- the images look pretty good.  Can you tell me more about
 the series termination effects?

 Again thank you for your help and I'll let the group know how it worked
 out.

 Alex

 --
 Dr. Alex Singer
 C.H. Best Institute
 112 College St. Room 70
 University of Toronto
 Toronto, Canada, M5G 1L6
 416-978-4033




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

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] stock solution of glutathione

2011-02-24 Thread Michael Thompson
Hi Rashmi,

The concentration of your stock solution will depend on how you plan to mix 
your screen conditions. It's not really that important as long as: 1.) 
Glutathione stays soluble, and 2.) You can make your condition with the desired 
glutathione concentration. If your glutathione is reduced you should probably 
make the stock fresh each time because reduced glutathione will oxidize, but if 
it is already oxidized it is probably ok to keep a stock for a little while. I 
would maybe keep it at 4 degrees...I'm not sure how well glutathione tolerates 
the freezer. If you need to make stock solutions that you plan to keep around 
for a while, remember that oxidative formation of disulfides is pH-dependent, 
with lower pH favoring the thiols (reduced) and higher pH favoring the 
disulfide (oxidized). This is because the thiol must be deprotonated before 
oxidation. If you're making a stock of reduced glutathione, I would recommend a 
pH of 6.5. For oxidized glutathione, I would recommend around 8.

Hope this helps,

Mike



- Original Message -
From: rashmi panigrahi rashmi.panigrah...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, February 22, 2011 11:42:15 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] stock solution of glutathione


Hi, 
I wanted to use glutathione for cocrystallizing with my protein at about 5mM 
concentration. What is the concentration of the stock solution to be prepared 
for crystallization studies. Do I have to make the stock solution of 
glutathione fresh each time or can I make the stock and store at -20degrees? 
Should I prefer to make the stock solution at a particular pH to prevent its 
oxidation?? 
suggestions awaited... 
-- 
rashmi 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] how to generate density map of selected residues

2011-01-14 Thread Michael Thompson
Hello Herman and others,

I am somewhat concerned after reading the following comment:

In pymol there is the infamous carve option, which can be used to select 
density for specific fragments and which can make bad density look good if you 
carve very tightly around your fragment.

I have been using PyMol to generate figures of electron density by creating 
.ccp4 format maps and displaying them in PyMol, using the option carve=1.6 as 
recommended by the PyMol Wiki. After reading Herman's reply, I am concerned 
that the figures I have been making don't accurately represent my density map. 
I haven't really found any information about what the carve option is 
actually doing. Can anyone comment on the proper way to display .ccp4 maps in 
PyMol so that they accurately depict the map without making bad density look 
good (or good density look bad for that matter)? Alternatively, is there a 
better method for making figures of density that does not involve PyMol, or 
eliminates any potential problems related to the carve option as Herman alluded 
to?

Thanks in advance for any advice,

Mike Thompson




- Original Message -
From: Herman Schreuder herman.schreu...@sanofi-aventis.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, January 14, 2011 7:08:48 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] how to generate density map of selected residues

Hi Jie,

What is the purpose of generating density of a selected fragment? In
pymol there is the infamous carve option, which can be used to select
density for specific fragments and which can make bad density look good
if you carve very tightly around your fragment.

I usually delete the residues of interest from the pdb and calculate an
omit map. The resulting difference (FOFCWT) map will show a relatively
unbiased density for the omitted residues.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of jy
Sent: Friday, January 14, 2011 2:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] how to generate density map of selected residues

Hi All,

I tried the mask map function in COOT to generate density map of
selected fragment. However, the map looked much better than the original
2Fo-Fc map. For example, weak density now became strong density.

I am wondering whether the mask map I generated using default is
actually a Fc map? is there a parameter setting to make it reflect the
true map?

Also, is there a way to select non-continuous residues other than a
segment? or what is a better program to do the job?

Thanks a lot!

Jie Yang

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] help with kinase purification

2011-01-03 Thread Michael Thompson
Hi Neeraj,

A few points to add to Artem's excellent advice:

If you would like to purify an untagged construct of your kinase, you could try 
an ATP affinity column. The caveats to this are 1.) the ATP resins can be 
expensive, and therefore it may be impractical to make a column with a high 
binding capacity, and 2.) Lots of proteins bind ATP, so your fractions might 
not be so clean (preceding your ATP column with an AS precipitation could help).

Artem mentioned that often eukaryotic kinases are expressed in insect cells, 
and also touched on the fact that they can be phosphorylated by other kinases 
within your expression system. It may be worth a try to see if you can get your 
protein to express in a bacterial system, because most bacteria (notably 
laboratory strains of E.coli) lack S/T/Y kinases. Autophosphorylation, however, 
cannot really be avoided. Certain competent E.coli are pretty good for 
expressing eukaryotic proteins. You might try the BL21 CodonPlus Rosetta strain 
(available commercially - novagen I think, or maybe Stratagene) because it has 
a plasmid that expresses a number of tRNAs that correspond to RILP codons that 
are common in eukaryotes, but rare in E.coli. 

Good luck,

Mike Thompson 


- Original Message -
From: Artem Evdokimov artem.evdoki...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Saturday, January 1, 2011 6:56:03 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] help with kinase purification


Each protein is different, but there are indeed similarities within classes. It 
can be dangerous/unproductive to generalize -- you will find out experimentally 
whether you can assume things or not. 

Depending on the expression method and the design of your construct you may or 
may not have a tag. Historically, a lot of eukaryotic protein kinases tend to 
be expressed in insect cells with a GST or His-GST tag. Protein kinases tend to 
favorably express in insect cells but the GST thing is in my experience more of 
a historical trend than a real requirement - I've expressed quite a few kinases 
with a simple His-tag. So, we can perhaps assume your first purification is a 
tag of some sort, and that you can cleave the GST away (His-tag typically does 
not have to be cleaved for crystallization, though it can certainly help). 
Depending on the purity and homogeneity of the enzyme you may want to try an 
orthogonal second step - a dye affinity resin or an ion exchanger, followed by 
size exclusion if necessary. If your kinase is autophosphorylated or if it's 
phosphorylated by another kinase you may have a mixed population of phosphates 
which can often be resolved by careful ion exchange chromatography (monoQ 
typically) either alone or in combination with phosphatase treatment. Note that 
phosphatases may not remove all phosphates from a mature folded protein - if 
you have phsophorylation and you *must* have a completely non-phosphorylated 
protein you may have to try co-expression with a suitable phosphatase. 

If your first purification step is 'generic' (like an AS cut or a crude ion 
exchange) nothing much changes for the following steps except you will have 
less pure starting material and therefore may have to do more total steps (and 
perhaps add another technique into the mix like HIC etc.). 

Good luck, 
Artem 

On Sat, Jan 1, 2011 at 1:03 AM, Neeraj Kapoor  nkap...@mail.rockefeller.edu  
wrote: 



Hi All, 
A very happy new year to everyone. I am beginning to purify a novel kinase and 
was wondering if there's a standard protocol / method that can be utilized to 
begin purification of a kinase. I am new to the field of kinases and would 
appreciate any help with respect to the precautions / pitfalls while dealing 
with kinase purification. 

Thanks and have a great year ahead! 
Neeraj 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate

2010-12-15 Thread Michael Thompson
I don't know how many of these crystals you have, but if you can spare one try 
freezing it straight out of the drop without cryoprotection. Certain salts can 
act as cryoprotectants at high enough concentrations. I don't know about 
phosphate salts, but I've had crystals that grew in 2.5M ammonium sulfate which 
I froze without cryoprotection and rarely saw an ice ring in my diffraction.

Good luck,

Mike


- Original Message -
From: Jerry McCully for-crystallizai...@hotmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, December 15, 2010 1:13:09 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium 
hydrogen phosphate

Dear All; 

Recently we got some crystals from the condition #51 in the SaltRx 
crystallization kit from Hampton research. 

It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test 
diffraction ASAP. 

What cryoprotectant did you use for this condition? 

Thanks a lot and have a nice holiday season! 


Jinghua 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Off Topic - Nickel Column

2010-11-17 Thread Michael Thompson
Dan,

You could try a denaturing purification to get rid of the binding partner.

First, take your cell lysate and spin it down at high speed to remove insoluble 
contaminants. (You probably do this already.) Then take your clarified lysate 
and dialyze it into buffer containing 6M Guanidine HCl. This will unfold 
everything, including proteases (no proteolysis under these conditions), and 
break the interaction between your protein and its binding partner. Then you 
can spin again at high speed to remove any additional aggregates and load this 
denatured sample onto the nickel column. The His-tag will still stick if the 
protein is unfolded in the presence of 6M GdHCl. Elute the protein in the same 
buffer with 6M GdHCl plus high imidazole concentration. Then you can take your 
eluent and dialyze out the GdHCl to refold the protein. 

Refolding (in the test tube, anyway) is not possible (practical?) for all 
proteins, but it often works very well.

Good luck,

Mike Thompson




- Original Message -
From: Daniel Bonsor bon...@bbri.org
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, November 17, 2010 8:49:37 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Off Topic - Nickel Column

I have a His-tagged protein which I am coexpressing with it's binding partner 
to prevent proteolysis. Once on the Nickel column I can remove 80% of the 
partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight. 
However the last 20% is difficult to remove, even if I reload the Nickel column 
and flush a further 2l of salt solution. I am wondering if I can increase the 
pH to 9.0 or 9.5. It should not effect the binding of His for the Nickel as the 
His-tag has to be deprotonated to bind, though will it causing stripping of the 
Nickel? 

Thanks


Dan

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] Crystal structures of phosphorylated proteins

2010-11-10 Thread Michael Thompson
Hi Sally,

Crystallization of proteins with multiple phosphorylation sites is often a 
difficult problem. Like you said it is often an issue of inhomogeneity. Several 
suggestions that may be helpful:

First, I would use a bit of your protein sample for Mass Spec analysis. It is 
possible (though maybe not likely if your protein has multiple sites) that your 
sample is homogenous and you don't even have to worry. 

Next, I would think about what you are trying to learn about this protein from 
the crystal structure. If you are interested in how the structure is effected 
by phosphorylation, there are several things you can do. To obtain a structure 
of the fully dephosphorylated protein you can use a nonspecific phosphatase 
before crystallization to remove all the phosphates. Alternatively you can 
mutate the phosphorylation sites. Mutating Ser to Ala, Thr to Val, and Tyr to 
Phe will probably not effect your protein's native structure very much (if at 
all), no matter how many substitutions you have to make. You should, however, 
determine the phosphorylation sites beforehand so you are not unnecessarily 
mutating S,T, and Y residues that aren't ever going to be phosphorylated. To 
learn how phosphorylation of Ser/Thr residues influences the structure, it is 
often useful to mutate the phosphorylation site to Glu, because it's size, 
shape, and charge properties are not very different from phosphoserine or 
phosphothreonine. This strategy is not perfect, but it can work well, 
especially since it directly addresses the problem of inhomogeneity.

A final suggestion is to just try and crystallize your protein with 
inhomogeneous phosphorylation. Depending on how inhomogeneous your sample is at 
each site, you will likely see phosphates that have only partial occupancies 
(and you might not ever see phosphates at sites that are weakly 
phosphorylated), but it still might be sufficient to gain the information you 
are interested in. Again, if you plan to do this, mass spec may help you 
understand your data. If you can mass spec digested protein you may be able to 
figure out which phosphorylation sites have high and low occupancy (I'm not a 
mass spec expert though), and you could compare this data with what you observe 
in your crystal structure.

Another quick thing to mention is to be weary of your expression system. If you 
express your protein in an exogenous expression system, the phosphorylation 
state you observe may be different (dramatically different in some cases) from 
what occurs in the native context, which is what you're interested in. Again, 
mass spec could help you here.

Hope this helps,

Mike Thompson





- Original Message -
From: Sally Pham Thanh Van sally.pha...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, November 10, 2010 11:03:49 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Crystal structures of phosphorylated proteins

To make it clearer, I'm looking for successful examples of
crystallization of phosphorylated proteins, especially ones containing
multiple phosphorylation sites, and would like to access
crystallizability of these protein types. I want to produce and
crystallize protein at its phosphorylation state but I learned
phosphorylation may cause inhomogeneity problem and interfere with
crystallization. It's not a good option to replace these sites anyway
because there are many and may affect native structure of the protein.

Any input would be appreciated,
Sally.







On Wed, Nov 10, 2010 at 1:26 PM, Atlanta Cook c...@biochem.mpg.de wrote:
 Perhaps you might pose a more directed question... Do you want to produce
 phosphorylated protein? Are you having trouble crystallising a protein that
 is phosphorylated?


 Atlanta




 On 10 Nov 2010, at 11:09, Sally Pham Thanh Van wrote:

 Dear all,

 Could you please tell me any information regarding to crystal
 structures of phosphorylated proteins? Your input would be
 appreciated.

 Best regards,
 Sally.



-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible Residues

2010-11-03 Thread Michael Thompson
Hi Buz,

Sorry to respond a little late, but if you are still tinkering with surface 
accessibility calculations, you can use the following web server that will 
calculate the diffusion accessibility of each atom in your input .pdb file:

http://nihserver.mbi.ucla.edu/diff_acc/

Diffusion accessibility is slightly different from surface accessibility, 
so depending on what you're interested in this may not be exactly what you 
want. From the web server:

Diffusion accessibility gives a measure of surface accessibility that is 
longer range in nature than ordinary solvent accessibility. The idea of 
diffusion accessibility was introduced several years ago (Yeates, TO (1995) 
Algorithms for Evaluating the Long-range Accessibility of Protein Surfaces. J 
Mol Biol 249(4): 804-15). Diffusion accessibility is a measure of how easily or 
frequently a specific point on a surface would capture a hypothetical diffusing 
(or randomly-walking) probe that is captured upon its first encounter with any 
part of a surface. Naturally, parts of a surface that are solvent-exposed in 
the traditional sense but lie in a deep surface depression will capture the 
probe only rarely, since the probe will more often encounter another part of 
the surface first. The results of a diffusion accessibility calculation can be 
useful in quantitatively evaluating potential binding regions of a protein 
surface, and are particularly useful in visualization.

The reason I still mention this is because diffusion accessibility vs. surface 
accessibility may be one way to answer your question of how to identify a 
surface residue vs. an interior residue. Also, the web server will output 
another .pdb file with the diffusion accessibility for each atom rewritten into 
the B-factor column. This is particularly useful for visualizing the 
surfaceness (as Ed put it) of each atom because you can open the output 
coordinates in PyMol and color atoms by B-factor which will give you a nice, 
colorful representation of the accessibility of the surface.

Hope this is helpful,

Mike 

- Original Message -
From: Buz Barstow b...@mac.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, November 3, 2010 6:55:54 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Software to Produce Linear Map of Surface Accessible 
Residues

Dear All,

Thanks for your suggestions! I ended up using the areaimol program in ccp4. 

My next question:

Given a list of accessible surface area, is there a generally accepted value of 
ASA above which a residue classifies as a surface residue, while below this 
number it is an interior residue?

Thanks! and all the best,

--Buz


On Nov 2, 2010, at 10:56 AM, Tim Gruene wrote:

 Hello Buz,
 
 I do not know what you mean by 'linear map', but according to its manual, the
 ccp4-program surface writes a list of accessible are per atom per residue,
 which you could convert into the total fraction per residue with not too much
 effort.
 
 Is this what you are looking for?
 
 Cheers, Tim
 
 On Tue, Nov 02, 2010 at 10:36:56AM -0400, Buz Barstow wrote:
 Dear All,
 
 I'm looking for a software program to produce, given a 3D atomic structure 
 of a molecule, a linear map showing the surface accessibility of residues in 
 a protein structure. 
 
 Would any one know of a program that can produce this sort of map.
 
 Thanks! and all the best,
 
 --Buz
 
 -- 
 --
 Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen
 
 phone: +49 (0)551 39 22149
 
 GPG Key ID = A46BEE1A
 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Reverse Translatase

2010-09-08 Thread Michael Thompson
Hi Jacob,

A couple more things to think about:

1.) How to get a Nobel Prize: Try an experiment if you have the means. 
Transform/transfect your favorite cell type with exogenous protein sequences. 
Sequence and see if they ever appear in the genome. Go after it...

2.) I realize that the idea of a reverse translatase doesn't necessarily 
imply that the sequence ever makes it into the genome, but if this is to be a 
truly useful biological system, it would need to include a reverse 
transcription and incorporation into the genome in order to pass the acquired 
information to daughter cells. Imagine the scenario in which the pathogen 
evades the immune system by copying host proteins. If they could not pass this 
information to daughter cells as they divide, those cells would instantly be 
susceptible to the immune system. There needs to be some selective pressure, an 
acquired benefit that can be passed to offspring, that would make this system 
truly biologically useful.

Now comes the question of how does this reverse translated/transcribed gene get 
into the genome. A transposase, sure, but how does this transposase know where 
to put the gene? It must be the appropriate distance from an appropriate 
promoter and enhancers, etc. otherwise it will not be expressed properly. And 
it has to have ribosome binding motifs (which would still be required even 
without genomic incorporation). Remember, the huge hurdle to much of gene 
therapy has been controlling where the exogenous gene inserts into the genome. 
Some incorporate in a highly nonspecific manner, in far too many places and/or 
interfere with other vital genes as they insert and in doing so cause a number 
of terrible side-effects like cancers, etc. 

3.) Codon bias has already been sufficiently explained by correlating codon 
usage with the expression/availability of their associated tRNAs. This has been 
proved experimentally, and I don't have a reference for this, but consider 
certain commercial competent cell strains for expression in E.coli. Cells such 
as BL21(DE3)-RILP are so effective in expressing proteins from different 
organisms from expression vectors, even without codon optimization for 
bacterial expression, simply because they contain plasmids that also 
overexpress certain rare tRNAs (for Arg, Ile, Leu, and Pro in the cell line 
mentioned), thereby increasing their availability and subsequent usage.

Keep chasing that Nobel Prize, but have a backup plan...you're clearly very 
creative.

Mike


- Original Message -
From: Jacob Keller j-kell...@fsm.northwestern.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, September 7, 2010 7:10:02 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Reverse Translatase

In terms of usefulness, I was actually thinking about cells learning how 
to make new proteins from other cells, or perhaps an immune system could use 
the info to make the right choice of starting materials. Also, codon bias 
could be explained as resulting from the nature of the reverse translatase 
machinery. Or an invader could copy the host's membrane proteins to evade 
detection. Ah, so many possibilities! And as I said before, considering that 
it would be so useful, and that the genius of macromolecular design observed 
in nature is apparently so unlimited, shouldn't it be out there somewhere? 
Nobel prize to the one who finds it...

Jacob

NB It should not cross our minds, I don't think, that if it were there, it 
would have been found. Small RNA phenomena, for example, went undetected for 
years, despite their commonness and high importance.


- Original Message - 
From: Artem Evdokimov ar...@xtals.org
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, September 07, 2010 8:29 PM
Subject: Re: [ccp4bb] Reverse Translatase


Regardless of whether a system like this exists in Nature or not -
it's fun to imagine!

On a microscopic scale one could propose a hypothetical mechanism by
which a completely unfolded polypeptide chain could be fed into a
gated (or state-locked) peptidase that may break the chain down in a
co-ordinated stepwise fashion; releasing individual aa's into some
sort of a nanoscale channel. The released aa's would then be
sequentially coupled to something resembling tRNA - with pre-formed
trinucleotides attached on the other end. Coupling would then
presumably permit the triplets to ligate to one another sequentially -
the resulting ssDNA or ssRNA would then have to be converted into a
stable ds-form via the usual means, or otherwise protected in one of
the usual ways. Codon space could be expanded by pre-loading carrier
molecules with more than one type of triplet per carrier (biased
towards whatever codon frequencies are prominent in the organism of
choice) although this in no way resolves the random nature of the
actual codon use within the resulting nucleotide sequence.

The issue of amino acid coupling selectivity is pretty hairy - the
best I could think of on a short notice is to have the 

Re: [ccp4bb] off topic-aggregation of proteins

2010-09-07 Thread Michael Thompson
Hi Bei,

I have fought this problem myself several times. In addition to some of the 
suggestions from Tim, here are some suggestions for additives:

As you mentioned Tween20 and Triton X-100 are very good for solubilizing 
difficult insoluble proteins. Those two are usually my first two weapons 
against insolubility.

NP-40 (this is a detergent, but as far as I know it's not available 
commercially anymore, but you can buy NP-40 substitute from several companies)

Glycerol (already mentioned)

Xylitol or Sorbitol (which also work well as cryoprotectants if your 
protein/crystals like(s) them)

Trehalose (+ or - extra phosphate in the buffer)

0.1M Arginine

Also, it's best to start with low concentrations of these additives and 
increase them as needed. As for working with detergents, if your protein is 
soluble and not a membrane protein, you want to be well below the CMC. Like 50% 
of the CMC, max. The idea is that you want the soluble detergent molecules to 
interact with the sticky parts of your protein, and if the detergent molecules 
are forming micelles they aren't doing their job very effectively.

Additionally, and I know this sounds a little insane - I was a skeptic at 
first, but others in my lab have had success with sonicating solutions of 
aggregated protein at very low intensity, very briefly. If your protein is 
reasonably stable but forms aggregates, I think this could work. (Like I said, 
I didn't beleive it at first, but CD scans of the sonicated protein show that 
it's still folded properly, and crystal structures have been solved from these 
samples.)

One last thing to mention, when you add some of these additives to your buffers 
they will become contaminated very easily. Those stubborn little microorganisms 
that manage to contaminate buffers as simple as tris and salt will LOVE to eat 
up some of these sugars, amino acids, and even detergents, so be extra careful 
about autoclaving bottles, filtering buffers, etc.

Good luck, as you are no doubt finding out this can be a difficult and 
frustrating problem.

Mike Thompson




- Original Message -
From: joybeiyang joybeiy...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, September 7, 2010 12:30:04 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] off topic-aggregation of proteins


Hi everyone, sorry for this none CCP4 question, but I am working on some tough 
proteins which easily got aggregated after purification, I am thinking of using 
some detergents rightnow, however, I am a newbie in this field, could you 
please give me some advice on the usage of detergents? 

here is some basic information about the protein: 
1. This protein is as big as 90KD. 
2. The productivity of the protein is very low, and the homogeneity of the 
protein is not good. Most of the proteins come out of the column at void 
volumn, and the rest of the proteins forms 3-4 peaks, and each of them features 
low peak height. 
3. I have already tried different truncates of the protein and homologous 
proteins from other species, up to now the protein on my hand is the best of 
them. 
4. I have stocks of the following detergents:n-Octyl-b-D, n-Decyl-b-D, 
FOS-Choline-12, n-Dodecyl-b-D, Cymal-5, CHAPS, Tween20, Triton-X100. 

my question is: 
1. would you please recommend some other detergents to try? 
2. should I try different concentration of the detergent and how? right now I 
just tried the concentration equals the CMC, some of the detergents do improve 
the homogeneity of the protein, however, once I concentrate the protein for 
crystallization, the detergent get concentrated too, and as you know that is 
very bad for crystallization. 
3. would you please recommend me some literature to resort to (about the same 
situation as mine or about the use of detergents)? 
4. any other suggestions or comments about how to improve the quality of the 
protein. 

Your suggestions and comments will help me a lot and will be highly 
appreciated. 

Many thanks to all of you! 

Bei 

2010-09-07 

joybeiyang 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Reverse Translatase

2010-09-06 Thread Michael Thompson
Jacob,

The idea is enticing, but don't forget that there are multiple degenerate 
codons for a given amino acid. Once the protein is synthesized, the specific 
codon information is lost.

I think that's a fundamental problem.

Keep the ideas coming,

Mike Thompson




- Original Message -
From: Jacob Keller j-kell...@fsm.northwestern.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, September 6, 2010 6:36:14 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Reverse Translatase

Dear Crystallographers,

does anyone know of any conceptual reason why a reverse translatase enzyme 
(protein--nucleic acid) could not exist? I can think of so many things for 
which such an enzyme would be helpful, both to cells and to scientists...! 
Unless there is something I am missing, it would seem to me conceptually 
almost impossible that it *not* exist.

Best Regards,

Jacob Keller


***
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
***

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] TLSMD Alignments

2010-08-24 Thread Michael Thompson
Hi All,

I have a question for those of you familiar with the TLSMD webserver. I am 
working on a structure with slightly imperfect 3-fold rotational NCS. My most 
recent .pdb file has been generated using Refmac (followed by a little 
tinkering in Coot), and during refinement I have been imposing medium main 
chain and loose side chain NCS restraints, and my R-factors don't really 
improve if I loosen the restraints further. This is the .pdb file I've also 
used an input to TLSMD.

The results of TLSMD do show that the residuals appear to slowly plateau when 
breaking the chains into 19 or 20 groups (all three A,B,C seem to converge 
similarly). When I look at the alignments, the TLS groups created for each 
chain do not align with each other well. The alignment of groups gets slightly 
better as more groups are added, which is partially just an issue of the groups 
being smaller and looking closer I think, but there is still significant 
stagger between neighboring groups. Is this typical for a structure with 
NCS-related chains? It seems somewhat counterintuitive to my understanding of 
how symmetric proteins should work (if the TLS motions reflect actual motions 
of the molecule). Perhaps the difference in TLS grouping between chains results 
from differences in Biso for NCS-related atoms that result from crystal 
packing? Maybe someone can shed some light on the situation?

Thanks a lot,

Mike Thompson




-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Disulfide Designer Program

2010-08-23 Thread Michael Thompson
I think that Disulfide by Design, or DbD, is geared toward monomeric proteins 
(ie. for enhancing stability). I'm sure you could get it to work with an 
oligomer with a little tinkering, but there is another server called sGAL that 
may be more like what you're looking for:

http://bioinformatics.oxfordjournals.org/cgi/content/abstract/22/24/3101

Also, I have designed intermolecular disulfide bonds using the following method 
(although it is a bit of a hacked up way to do things, but it worked well in my 
situation). Put your PDB files for each chain of the oligomer through the 
following diffusion accessibility server:

http://nihserver.mbi.ucla.edu/diff_acc/

This server will take your PDB file and rewrite the B-factor column with values 
for the diffusion accessibility of each atom. Now you can look at your PDB 
files in PyMol, and if you choose color by B-factor you will actually be 
coloring the atoms by diffusion accessibility. Do this for each chain of the 
oligomer separately. Now take those individual chains, colored by diff. 
accessibility and overlay them onto the structure of the oligomer. Look for 
residues whose C(gamma) are both accessible at the surface of each chain, and 
are within reasonable distance for a disulfide bond. You can check this by just 
measuring the distance, or if you want to be more precise, you can take several 
ideal disulfide bonds from other structures and compare the distances (and 
dihedral angles) by overlaying them onto your selected residues. If you find a 
good set of residues that match all the criteria I listed, chances are they 
will be good candidates to disulfide bond with each other if mutated to Cys. 
Remember that with disulfide bonds, geometry (dihedral angles) is very 
important in addition to the bond length. I don't recall off the top of my head 
what values for the dihedral angle and bond length are ideal, but this info is 
readily available in the literature. And also, your bond will never form if the 
gamma position of the side chain is buried by surrounding residues (thus, the 
use of the diff. accessibility server - I learned this the hard way by making 
many unsuccessful mutants). Again, this is kind of a drawn out way of doing 
things compared to just throwing your PDB file at a server like sGAL, but I 
feel that it is a bit more rigorous, and it has been successful in my hands.


Good Luck,

Mike Thompson



- Original Message -
From: Konstantin v. Korotkov k...@u.washington.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, August 23, 2010 4:09:50 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] Disulfide Designer Program

Hello,

You could try Disulfide by Design:
http://cptweb.cpt.wayne.edu/DbD/

Good luck,
Konstantin

On Mon, 23 Aug 2010, Jacob Keller wrote:

 Dear Crystallographers,

 I remember having heard of a program which takes a given oligomeric assembly, 
 and suggests optimum disulfides to stablize the complex. Can someone refresh 
 my memory which program that is, and where it is available?

 Best Regards,

 Jacob Keller

 ***
 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
 ***


--
Konstantin Korotkov, Ph.D.

Research Scientist
University of Washington
Department of Biochemistry
Box 357742
Seattle, WA 98195-7742

(206)616-4512
k...@u.washington.edu
--

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


[ccp4bb] Refinement/Model-Building Density Modification

2010-08-18 Thread Michael Thompson
Hello All,

I am currently solving a structure at 2A resolution with phases obtained from 
molecular replacement. Using the MR solution, I began refinement with Refmac 
using NCS restraints. I am currently building the parts of the model that were 
left out of the MR search model, and have just about successfully completed all 
three NCS-related chains. Obviously I will continue to use the most complete 
model for refinement, and plan to release the NCS restraints over the parts of 
the molecule that don't quite seem to obey perfectly. 

My question is, once I have connected density for all three chains will it 
still be worthwhile to perform density modification, such as solvent 
flipping/flattening or histogram matching (implemented through SOLOMON and/or 
DM) to improve phases? It seems that I have always been told that density 
modification is typically carried out at the beginning of refinement, prior to 
any model building, however my understanding of these types of density 
modification (particularly solvent flipping/flattening) leads me to believe 
that they would be most effective when more of the molecular envelope can be 
identified, such as during later stages of refinement.

Also, I read something recently that lead me to believe that the solvent 
flattening procedure may be implicit in the implementation of NCS averaging in 
refinement software. I understand that the two processes are fundamentally 
different and independent of one another, but the information I recently read 
described something like the following (unless I misinterpreted). Because real 
space NCS averaging requires identification of the molecular envelope in the 
same fashion as solvent flattening, during NCS averaging the envelope is 
identified then the map is solvent-flattened and averaged using the NCS 
operator. I am unfamiliar with the inner-workings of most crystallographic 
software, so I was wondering if this is how NCS averaging is implemented in 
Refmac? I suppose another way to ask the question would be: If I have an 
NCS-averaged map from Refmac, is it already solvent-flattened?

Any help would be much appreciated. I am still relatively new to the refinement 
process.

Thanks,

Mike Thompson


-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Lactose refinement

2010-08-16 Thread Michael Thompson
Hello Rex,

Just a few biologically-related ideas that may support modeling the planar 
conformation.

I'm not sure if the protein you're working with has enzymatic activity, but is 
it possible that the ring strain is indeed real and may be a part of the 
reaction mechanism? Are any parts of the protein in contact with the flattened 
ring in such a way that they might be pushing or pulling the ring into the 
planar conformation? 

Best,

Mike




- Original Message -
From: Rex Palmer rex.pal...@btinternet.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, August 16, 2010 9:44:39 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Lactose refinement


We are attempting REFMAC refinement of a protein structure which is complexed 
with lactose. One of the rings (the one oriented away from the protein) loses 
the chair conformation which is flagged as a problem by COOT check chiral 
volumes . 
Is there anything we can do to restrain the ring as a chair and would this 
necessarily be a valid move? ie why can't the conformation deviate from the 
norm? 

Rex Palmer 
Birkbeck College 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] monomer-dimer

2010-08-11 Thread Michael Thompson
I agree completely with Anastassis that the equilibrium will be effected by 
changing the concentration of the sample during gel filtration, however I 
wanted to point out that the elution volumes of the two species are independent 
of their populations. I apologize if I was misleading.

Mike


- Original Message -
From: Anastassis Perrakis a.perra...@nki.nl
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, August 11, 2010 2:10:16 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] monomer-dimer

Dear all, 


If I may add that I find the statement 


First, remember that gel filtration elution volumes are independent of 
conditions like flow rate and protein concentration (unless there are 
nonspecific interactions at high concentration), but like I described before 
temp is a factor. 


a bit misleading. While concentration will not change where the monomer or the 
dimer appears in the elution volume, concentration will affect the 
monomer-dimer equilibrium during your gel-filtration run. 


Thus, I would say that concentration is a factor. If your dimer has a kD of 
~100uM, and you inject it at a concentration of ~100uM, after getting diluted 
during gel-filtration (about ten-fold) it will appear 90% as a monomer ... The 
results of any analytical technique to determine stoichiometry are 
concentration dependent, and concentration is actually the major variable that 
needs to be considered to define the oligomerization state (in AUC this can be 
done nicely). And do not forget that the in-vitro oligomerization state does 
not necessarily imply the same for in vivo, so please do make mutants to prove 
it before submitting the paper ... 


A. 



On Aug 10, 2010, at 1:38, Bostjan Kobe wrote: 



Dear Intekhab 

Let me just add to this that gel filtration is not an accurate method for 
determination of molecular mass, because the migration on the column depends 
on the shape of the protein. 

The following methods can be used to determine molecular mass irrespective 
of shape: 
- MALLS (multi-angle laser light scattering or static light sxattering) 
- sedimentation equilibrium on analytical ultracentrifuge (AUC) 
- native mass spectrometry 

For a short recent review on issues associated with determining oligomeric 
state from crystal structures, with older references and relevant 
bioinformatic tools cited in there, please see 
http://www.ncbi.nlm.nih.gov/pubmed/19021571 

Bostjan 


On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: 



To determine the oligomeric state of a protein (monomer or dimer in your 


case), it's useful to use the PISA server. You upload your pdb file from 


the crystal structure.The server calculates the areas of interfaces 


(buried area) and deltaG (change in Gibbs energy) upon oligomer 


dissociation. (E. Krissinel and K. Henrick (2007). /Inference of 


macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 


774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein 


Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 


2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. 


E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. 


J. Comp. Chem., in press; published on-line 6 May 2009; DOI 


10.1002/jcc.21303} 


If the interface area (divided by 2 per one protomer) is greater than 


1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's 


a dimer. However, don't forget that most dimers can dissociate into 


monomers upon dilution. There is a dynamic equilibrium between dimers 


(oligomers) and monomers that depends on their concentration and the Kdiss. 


Separating them in any method will disturb this equilibrium. If the 


re-equilibration time is greater than the separation time, you can see 


both monomers and dimers. You can even roughly calculate the 


dissociation constant: 





Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you 


an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of 


dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, 


protein needs to dissociate easily for the biological function. 





Maia 





intekhab alam wrote: 




Hi everyone 




Sorry for some non specific query! 









i am working with a protein that shows a dimer in the crystal 




structure but when i tried to figure out that with standard molecular 




markers in gel filteration (superdex-200, 24ml column) it turned out 




to be a monnomer. Native gel analysis after incubating the protein at 




20 degree, 37 degree showed more dimer at 20 degree celcius as 




compared to 37. I tried similar strategy in gel filteration by 




incubating my protein at various temperature,where a lot of 




precipitation was observed at 37 degree celcius and after removing the 




precipitates i run the gel filteration that has 0.5 ml higher elution 




volume as compared to samples incubated at 20 

Re: [ccp4bb] monomer-dimer

2010-08-09 Thread Michael Thompson
Hello Intekhab,

Your results do not seem surprising at all. It is not uncommon for molecular 
interactions such as dimerization to be more stable at lower temperatures, and 
this is exactly why you are seeing the shift to higher elution volumes at lower 
tempratures. At lower temperatures, both the monomer and dimer are likely to be 
more compact in solution due to less thermal fluctuations in the overall 
structures. Remember that protein structures are always in motion, and lowering 
the temperature restricts these motions, and therefor lowers the effective 
radius of the molecule in solution as it moves through the column. And of 
course smaller molecules elute at higher volumes, so this probably explains 
what you see in the cold room.

As for some of the other concerns you have with your gel filtration 
experiments, I can offer the following suggestions. First, remember that gel 
filtration elution volumes are independent of conditions like flow rate and 
protein concentration (unless there are nonspecific interactions at high 
concentration), but like I described before temp is a factor. That being said, 
often analytical gel filtration experiments are more informative at moderate 
concentrations instead of high concentrations, because this will favor the 
formation of relevant oligomers, instead of oligomers and aggregates that form 
only at high concentrations and aren't really biological. When you do your 
incubation experiments, try using lower protein concentrations or shorter 
incubation times. This might prevent the formation of precipitates and will 
give you more biologically relevant information - after all, most proteins are 
not available in the cell at very high concentrations, so if your dimer is 
biological, the kd is likely pretty low. Also you could try another experiment 
like a pulldown with tagged/untagged constructs, or SPR. These experiments (SPR 
particularly) would also tell you if the kd is reasonable for a biologically 
relevant interaction. One more thought is to be sure your protein is not 
degrading at high temperatures, which may be the reason your 37 degree 
incubation results in increased elution volume. Mass spec could help you here.

Finally, to determine the biological relevance of your dimer you should do an 
analysis of the dimer interface seen in the crystal structure. I believe that 
for the average biological oligomer, the oligomerization interface buries 
approx 1200-2000A2 of surface area, whereas the average crystal contact buries 
approx 400-800A2. Some older work related to these analyses has been published 
by Joel Janin and Janet Thornton. Also, some webservers like PISA attempt to 
predict the relevant oligomerization states of proteins in the PDB based on 
interfaces seen in the crystal structures. You might look there for a good 
method.

Good Luck,

Mike Thompson




- Original Message -
From: intekhab alam faisal...@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, August 9, 2010 4:37:45 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] monomer-dimer


Hi everyone 
Sorry for some non specific query! 

i am working with a protein that shows a dimer in the crystal structure but 
when i tried to figure out that with standard molecular markers in gel 
filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native 
gel analysis after incubating the protein at 20 degree, 37 degree showed more 
dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel 
filteration by incubating my protein at various temperature,where a lot of 
precipitation was observed at 37 degree celcius and after removing the 
precipitates i run the gel filteration that has 0.5 ml higher elution volume as 
compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is 
this significant) 
Furthermore i have done some experiments in cold room (4 degree) where the 
elution volume is stuck at a point irrespective of the conditions (as Flow 
rate, concentration of protein etc) and that is higher than that of the room 
temperature by 1 ml. 
Standard moleculr weight markers also show higher elution volume in cold room 
in comparison to the room temperature by 1 ml. 

I will be highly obliged if someone suggest some literature or any otherway to 
do gel filtrtaion so that i can clearly resolve this issue. Also let me know if 
there is some literature available on effect of temperature on the elution 
volume of proteins. 

Thanks in advance 

-- 
INTEKHAB ALAM 
LABORATORY OF STRUCTURAL BIOINFORMATICS 
KOREA UNIVERSITY, SEOUL 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu


Re: [ccp4bb] Quality of His-Select Resin After Regeneration

2010-07-22 Thread Michael Thompson
Hello Matthew,

I almost always charge my IMAC resin with Co instead of Ni. I find that it 
gives much better purity because the selectivity is better. This is because the 
coordination sphere around the Co atoms require more His residues than the Ni 
coordination sphere. I believe Co requires 4 His, while Ni only requires 2. 
That being said, although it is more selective, proteins with a His tag are 
likely to have lower affinity for Co, and will therefore elute in lower 
imidazole concentrations, and if your His tag is not entirely exposed, you may 
have issues getting it to bind at all. I have never had this problem, but I 
have heard that it can be an issue. It is probably worthwhile to try cobalt, 
but do it under the assumption that there is a small chance you will have to go 
back to Ni.

Also, if you use cobalt, you may notice a slight color change in the column 
when you run it under certain buffer conditions. Some additives cause the color 
to change from light pink to a purplish color. This is ok, the column will 
still bind your protein fine. Just be sure to look in the resin manufacturer's 
manual to make sure you don't have any incompatible components in your buffer. 
In general, anything that can go over the column when it is charged with Ni 
should be ok for Co as well.

Good luck,

Mike Thompson







- Original Message -
From: Matthew Bratkowski mab...@cornell.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, July 22, 2010 7:05:21 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] Quality of His-Select Resin After Regeneration

Hi. 

We have been using His-Select Resin from Sigma in our lab for a number of years 
now. When we first bought the resin, I usually got much better purity of His 
tagged proteins compared with regular Ni-NTA resin. However, after regenerating 
the resin several times, the level of purity seems to have declined. Has anyone 
else noticed this with His Select? In general, could someone suggest the 
typical lifespan of His Select or Ni2+ resin in general? What about 
Glutathione resin? 

I was also wondering if anyone had experience using cobalt resin? What is the 
binding capacity of cobalt compared to nickel, and is the selectivity any 
better than either His-Select or regular Ni-NTA? Also, is it possible to just 
strip nickel off of the resin and then recharge it with cobalt? 

Thanks, 
Matt 

-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu