Re: [ccp4bb] Efforts been made to solve protein (soluble) aggregation

[Bosch, Juergen]

Co expression of binding partner perhaps ? And then tagging the other protein 
partner ?
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://lupo.jhsph.edu

On Mar 6, 2014, at 2:13, "WENHE ZHONG"  wrote:

> Dear CCP4 friends,
> 
> I have been searching around the CCP4 mails for the discussion of soluble 
> protein aggregations, as I have the same problem recently. Many efforts I 
> have tried but without success. Here, I would like to summarise what I have 
> done and maybe any of you come across some ideas.
> 
> 1. My protein (not membrane protein) is around 35kDa with a pI of 8.6. It was 
> expressed in E.coli as a fusion protein where its N-ter was fused by MBP-tag 
> with a flexible linker (Factor Xa cleavage site available). The whole fusion 
> protein is ~77kDa (pI 6.1). 
> 2. After MBPTrap affinity purification, the protein was run through 
> gel-filtration column it came out at void volume. The aggregation was also 
> confirmed by DLS. The buffer condition is: 20 mM HEPES, pH7.4, 125 mM KCl, 
> 20% glycerol, 40 mM maltose, 5 mM DTT.
> 3. I made several truncations and only one gave me a monomer (The other 
> truncations were all soluble aggregates). However, this monomeric truncation 
> is small (~5 kDa) and not important for the function (not interesting 
> fragment). At least, this monomeric truncation suggests that the aggregation 
> is not due to the aggregation of MBP. The truncations were designed in both 
> two versions: long linker (including TEV cleavage site) and short linker 
> (-Ser-Ser-Ser-).
> 4. Hampton detergent screens and high salt (1-2 M KCl or NaCl) were tried on 
> both truncations and full-length protein, but none of them work (DLS 
> confirm). Divalents and other ions should not bind to this protein. 
> 5. The full-length aggregated protein is still functional in Gel-shift assay, 
> which indicates the protein is corrected folding.
> 
> I guess "soluble aggregation" is a common problem for tough proteins. If 
> anyone has experience on it and can share with us, please drop an email.
> 
> Kind regards,
> Wenhe

Re: [ccp4bb] Peptide solubility issues

[Bosch, Juergen]

For that purpose we’ve developed this expression system that may or may not be 
useful to your specific problem:
http://www.ncbi.nlm.nih.gov/pubmed/23996492
J Mol Recognit. 2013 
Oct;26(10):496-500. doi: 10.1002/jmr.2292.
Development of a multifunctional tool for drug screening against plasmodial 
protein-protein interactions via surface plasmon resonance.
Boucher 
LE1,
 Bosch 
J.

Sorry, for the self advertisement (at least it’s not attached), but perhaps it 
is a useful tool for others as well.

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Mar 5, 2014, at 8:15 PM, Mo Wong 
mailto:mowon...@gmail.com>> wrote:

Many thanks to all the people who contacted me. I actually went rogue and 
decided to initially reconstitute in 10mM glycine, pH 9.5 to 2 mg/mL, sonicate 
for a few seconds and then immediately add an equal volume of more concentrated 
neutral pH buffer to minimize any possibility of alkaline hydrolysis. The 
peptide solubilized nicely, and the SPR sensorgrams are much more promising. My 
only concern is if glycine might interfere with peptide binding (I suspect not, 
but please let me know if you think otherwise) - a control doesn't suggest 
glycine likes to stick to the protein surface.

A few people pointed me to links on solubilizing lyophilized peptides; however, 
the one I had previously been consulting is, in my opinion, the most thorough:

http://www.bachem.com/service-support/faq/handling-of-peptides/

Thanks again.





On Wed, Mar 5, 2014 at 6:52 AM, Toufic El Arnaout 
mailto:elarn...@tcd.ie>> wrote:
Hello Mo Wong,
Some points below are good, but don't underestimate custom peptides 
sometimes... can be much harder/expensive than recombinant proteins.
If you ordered the peptides it is good to know how they synthesized them, and 
how the elution profiles during purification (RP HPLC..) looked like, 
particularly that they are very hydrophobic. Did they use formic acid, or maybe 
they already dissolved them in DMSO.. Did they verify the product/bonds with 
MALDI-TOF for example and NMR?
Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final 
concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 
% solvent at 1 mM..
Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), 
some detergents and even a minor DMSO/ethanol can affect the measurements 
drastically.
Best wishes

toufic el arnaout




On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong 
mailto:mowon...@gmail.com>> wrote:
Hi all,

Slightly off topic - but I'm having trouble solubilizing some peptides for SPR 
and hoped someone on the BB might have some other suggestions.

The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 
residues are hydrophobic and 2 are acidic. Peptides have been tested with and 
without N- and C-terminal modifications (amidation/acetylation).

I have tried:
ddH2O
raising (and lowering) pH (tested up to 8.5) with different buffers
Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine 
present in the peptide) -  peptide still visibly precipitates out at 100uM in 
5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt)
Adding a trace amount of detergent (0.005% Tween 20)

I'm guessing I could try other co-solvents such as ethanol or initially 
solubilizing peptide in dilute NaOH before bringing the pH down with addition 
of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd 
rather have some insight from people before I waste any further peptide.


Thanks for any suggestions.

Re: [ccp4bb] How to find the unfound part of a big protein

[Bosch, Juergen]

Hi Bingfa,

first suggestion would be to process your data with the anomalous flag turned 
on - just in case you happen to have some  metal bound coincidentally and you 
happen to have collected at a decent wavelength to pickup some anomalous 
scattering. ~30% of proteins in the PDB have a metal bound just FYI and not all 
of them were soaked in on purpose.

In case you should be that lucky, then you should be able to use the HA phases 
+ your MR model to help building.

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 13, 2014, at 4:53 PM, Sun Bingfa 
mailto:sunbin...@gmail.com>> wrote:

Dear Crystallographers,

I'm working on a ~90KDa membrane protein, with big extracellular part, probably 
function as dimer.
Now we have dataset to ~4.2 Angstrom and using extracellular homolog structure 
we can find a solution for this part(~45% of the whole molecule MW) through 
molecular replacement, and the molecules are packed as layers, and the other 
part are presumably between these layers. However, we are having trouble to fit 
the rest of the protein even though there're some density between the solved 
part. Rfree is at 40% now.

We're trying to do heavy atom soaking, such as TaBr. We collected data for MIR 
but it's not helping so far. (Can I combine these MIR data with the native 
dataset because the MIR set is only at ~6.5 Angstrom)?

Other information: this protein is expressed in Sf9 cells (so very hard to do 
Se-Met derivatives). The crystals is nice and big and cubic.

Any suggestions or examples? Thanks a lot.

Bingfa

Re: [ccp4bb] Sister CCPs

[Bosch, Juergen]

I guess somebody could add it here perhaps ?
http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Special:Allpages

or start their own wiki on "Tips & tricks in crystallography" and why you 
should always have olive oil when traveling to a synchrotron (well nowadays 
it’s becoming more difficult to actually be physically present at such a 
facility, then the olive oil won’t help either for cryo protection)

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 13, 2014, at 10:21 AM, Nat Echols 
mailto:nathaniel.ech...@gmail.com>> wrote:

One comment (not a complaint) on all this: it seems like the same questions get 
asked over and over again.  If there is a good place for a general 
crystallography FAQ list it is well past time for one to be put together - or 
maybe it just needs to be better advertised?  At a minimum, for instance:

- what cryoprotectant should I use?
- how do I get big single crystals?
- how do I improve diffraction?
- how can I tell if I've solved my structure?
- why is my R-free stuck?
- is  suitable for publication?

Some of the other common queries ("name my blob!") still need to be handled on 
a case-by-case basis, but it would be much more efficient for everyone if the 
standard answers were collected somewhere permanent.

-Nat



On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov 
mailto:eugene.val...@gmail.com>> wrote:
I absolutely agree with Juergen.

Leaving aside methods developers, who are a completely different breed, there 
is no such thing as a "crystallographer" sitting in a dark room solving 
structures all day. If there are, these are anachronisms destined for 
evolutionary demise.

More and more cell biologists, immunologists and all other kinds of biologists 
are having a go at doing structural work with their molecules of interest 
themselves without involving the "professionals". Typically, they learn on the 
job and they need advice with all kinds of things ranging from cloning and 
protein preps through to issues with tetartohedrally-twinned data and 
interpreting their structures.

So, a modern structural biologist is one who is equipped for the wet lab and 
has some idea of how to go about solving structures. CCP4BB is a wonderful 
resource that is great for both the quality of the advice offered to those that 
seek it and for the variety of topics that are addressed in the scope of 
structural biology. I have learnt greatly from reading posts from very skilled 
and knowledgeable scientists at this forum and then implemented these insights 
into my own research. I am very grateful for this.

In short, please do not discourage your colleagues, particularly very junior 
ones, from posting to the CCP4BB. Some of the questions may appear quaint or 
irrelevant but it is easy to simply ignore topics that are of no interest!

Eugene


On 13 February 2014 14:41, Bosch, Juergen 
mailto:jubo...@jhsph.edu>> wrote:
Let me pick up Eleanor’s comment:
is there something like a crystallographer today ? I mean in the true sense ?
I think as a “crystallographer” you won’t be able to survive the next decade, 
you need to diversify your toolset of techniques as pointed out in this article
http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a

And I’m not quite sure how software developers see themselves, as I would argue 
they are typically maybe not doing so much wet lab stuff related to 
crystallography (I may be wrong here) but rather code these days.

What “type” of crystallographer is a software developer ?

I think like our beloved crystals “we” come in different flavors. And we need 
to train the next generation of students with that perspective in mind.

Just my two cents on a snowy day (>30cm over night)

Jürgen
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu<http://lupo.jhsph.edu/>

On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:

I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:
It's not an e-mail bulletin board, but Researchgate seems to be quite
popular for wet lab questions. IMO the Q&A section of the social network is
a bit messy. That said, the quality seems to improve gradually.

Cheers,
Robbie

Se

Re: [ccp4bb] Sister CCPs

[Bosch, Juergen]

Let me pick up Eleanor’s comment:
is there something like a crystallographer today ? I mean in the true sense ?
I think as a “crystallographer” you won’t be able to survive the next decade, 
you need to diversify your toolset of techniques as pointed out in this article
http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a

And I’m not quite sure how software developers see themselves, as I would argue 
they are typically maybe not doing so much wet lab stuff related to 
crystallography (I may be wrong here) but rather code these days.

What “type” of crystallographer is a software developer ?

I think like our beloved crystals “we” come in different flavors. And we need 
to train the next generation of students with that perspective in mind.

Just my two cents on a snowy day (>30cm over night)

Jürgen
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 13, 2014, at 6:41 AM, Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:

I agree with Frank - it keeps crystallographers modest to know how
challenging wet lab stuff still is..
Eleanor

On 12 February 2014 19:23, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:
It's not an e-mail bulletin board, but Researchgate seems to be quite
popular for wet lab questions. IMO the Q&A section of the social network is
a bit messy. That said, the quality seems to improve gradually.

Cheers,
Robbie

Sent from my Windows Phone

Van: Paul Emsley
Verzonden: 12-2-2014 19:23
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: Re: [ccp4bb] Sister CCPs


On 12/02/14 15:59, George Sheldrick wrote:
It would be so nice to have a 'sister CCP' for questions aboud wet-lab
problems that have nothing to do with CCP4 or crystallographic
computing, The is clearly a big need for it, and those of us who try
to keep out of wet-labs would not have to wade though it all.


FWIW, the remit of CCP4BB, held at jiscmail-central, is describes as:

/The CCP4BB mailing list is for discussions on the use of the CCP4
suite, and macromolecular crystallography in general./



Thus wet-lab questions are not off-topic (not that anyone recently
described them as such).

Having said that, Jiscmail mailing lists are easy to set-up (providing
that you can reasonably expect that the mailing list will improve
knowledge sharing within the UK centered academic community) and
relatively low maintenance. I, for one, would not be entirely unhappy to
miss out on questions about lysis.

Paul.

Re: [ccp4bb] off-topic: bug busting

[Bosch, Juergen]

If heat is a concern, you can place your Emulsiflex into a bin with lots of 
ice. We have the version with cooling after lysis, but if you are paranoid you 
can cool the whole thing.
It has the footprint of a 15” MacBook Pro but it’s way more expensive than that 
:-)

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 5, 2014, at 11:49 AM, Michael C. Wiener 
mailto:mwie...@virginia.edu>> wrote:

Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics 
cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a 
shared-use M110-P Plug and Play is used by most of the membrane-heads at my 
place. I've generally been happy (or at least not unhappy) with it.

However, we've been having some QC issues with a membrane protein that we're 
making in S. ceresvisiae (Sc), and I'm having some concerns about sample 
heating. Can anyone comment on Microfludics vs Avestin vs Constant Systems vs 
Retsch vs whatever-else for cracking Sc cells? These days, we're working up 
~300-400g of paste at a time.

Thank you very much!

-MW

Michael C. Wiener, Ph.D.
Professor
Department of Molecular Physiology
and Biological Physics
University of Virginia
PO Box 800886
Charlottesville, VA 22908-0886
434-243-2731
434-982-1616 (FAX)

On Wed, 5 Feb 2014 00:34:11 -0500
Anirban Banerjee mailto:ani...@gmail.com>> wrote:
I will be curious to know about people's experiences with membrane
proteins and lysing yeast cells with the Microfluidizer and how that
compares with using a Retsch Miller, i.e. grinding in a liquid
nitrogen cooled stainless steel chamber and  plunging in liquid
nitrogen in between grinding cycles.

I am worried that the Microfluidizer is not as mild w.r.t. heating as
they claim it to be. That would, of course, perfectly qualify as my
OCD.

Any insights will be really appreciated.

Thanks,

Anirban

On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin 
mailto:mfrank...@nysbc.org>> wrote:
Hi Phoebe -

"Cost-effective" may not be the applicable word here, but the Microfluidizer
works very well:

http://www.microfluidicscorp.com/index.php?option=com_content&view=article&id=19&Itemid=76

This gadget runs on house compressed air (don't try to use a compressed air
tank - you'll empty it in minutes).  It's a bit noisy, but so is a
sonicator.

The Microfluidizer really shines with large volumes of lysate - like 1 L and
up.  If you're only processing 100-200 mL at a time, I think sonication is
the best way to go.

Hope that helps,
Matt



On 2/4/14 11:49 AM, Phoebe A. Rice wrote:

Some time ago, there was a nice discussion of cost-effective, wimpy
protein-friendly ways to break open E. coli.  We're thinking about replacing
an aging sonicator.  If people have a favorite gizmo, could they repeat that
advice?
thank you,
 Phoebe Rice

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp



--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374

Re: [ccp4bb] off-topic: bug busting

[Bosch, Juergen]

Emulsiflex C5
alternative freeze-thawing, the old way we used to do this spiking with some 
Lysozyme
or grinding it in a LN2 cooled mortar, is very effective and pretty cost 
effective, assuming you don’t pay for the LN2

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 4, 2014, at 11:49 AM, Phoebe A. Rice 
mailto:pr...@uchicago.edu>> wrote:

Some time ago, there was a nice discussion of cost-effective, wimpy 
protein-friendly ways to break open E. coli.  We're thinking about replacing an 
aging sonicator.  If people have a favorite gizmo, could they repeat that 
advice?
thank you,
  Phoebe Rice


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] identifying protein crystals via visible light only?

[Bosch, Juergen]

[Advertisement on] http://www.janscientific.com/#!/gallery [Advertisement off]

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 4, 2014, at 10:10 AM, Richard Gillilan 
mailto:r...@cornell.edu>> wrote:

Some years ago, I remember hearing about a microscope that used *visible* light 
combined with some proprietary image processing algorithm to
distinguish between protein crystals, salt, and background. I can't remember 
the company name or researchers involved.

Has anyone here heard of this?


Richard Gillilan
MacCHESS
Cornell

Re: [ccp4bb] Isolation of protein-protein complexes.

[Bosch, Juergen]

Hey David,

the 1 nM Kd by SPR sounds fishy to me - did you do these measurements yourself ?
Unless this is an antibody-protein complex or nanobody-protein complex or 
protein-small molecule complex, the value is too low (for a normal PPI, I would 
expect  >50 -500 nM). Possible explanation for the SPR result is non-specific 
binding of the ligand (your protein in solution) to the chip.
Which would in turn also explain your negative result in SEC eventually (what 
SEC material did you use ? Is it at the pH negatively charged perhaps - like 
the SPR chip).
Are you running the SEC in the same buffer as used in either ELISA, SPR or DPI ?
Most likely the main difference between those experiments and yours is the 
temperature, as I assume you ran your SEC at 4˚C.
Just a thought.

Jürgen


On Jan 21, 2014, at 10:51 AM, David Briggs 
mailto:drdavidcbri...@gmail.com>> wrote:

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple groups, and by 
multiple techniques - namely ELISA, SPR and DPI.

The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, and as a 
first step I attempted to purify the complex by mixing the two proteins (same 
protein preps and same buffers as the SPR experiment) and then running them 
down a gel filtration column (Superose 6 - predicted size of the complex is 
~500kDa).

Somewhat irritatingly the two proteins separate beautifully on the column into 
two distinct peaks. There is no trace of complex formation when the peaks are 
analysed by SDS-PAGE.

As far as I am aware, two proteins that interact this strongly should remain 
associated during gel filtration, and I was wondering if anyone else has 
encountered anything similar in the past, and if they managed to resolve the 
problem, how they went about it?

Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] How to show the ligands (both as sticks and sphere) as shown here in Pymol

[Bosch, Juergen]

Hi Wei,
make two objects of your ligand, display one as sphere and the other as stick. 
Then change the transparency setting for the spheres.
Jürgen

On Jan 19, 2014, at 11:40 PM, Wei Shi 
mailto:wei.shi...@gmail.com>> wrote:

Hi all,
Please see attached Fig where they show the ligand both as sticks and spheres 
at the same time. Does anyone happen to know how to display the ligand like 
this? Thank you so much!

Best,
Wei




..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Protein concentration form chromatograms

[Bosch, Juergen]

Step1:
visit Protparam tool and C&P your sequence, scroll down until you find the 
extinction coefficient part. If OD280 is not close to 1 then make sure to take 
that into account in your chromatogram say it is 0.7
step2: look at your mAU's
If your peak is 1000 mAU then you have 0.7 mg/ml in that fraction
Step3: you are pretty lazy
google for it

Jürgen

On Jan 15, 2014, at 10:09 AM, Karel Chaz 
mailto:karel.c...@gmail.com>> wrote:

Dear all,

A question for the biochemistry-inclined folks in the bb; how do I calculate 
protein concentration of chromatography fractions, starting from Abs280 from 
the UV monitor? I know I could figure it out myself if I really tried, but why 
bother when I have access to so many brilliant minds


Thanks to all,

K

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Heavy atom sites

[Bosch, Juergen]

Didn't mlphare use to print those values in the log file ?
Jürgen

On Dec 15, 2013, at 4:29 PM, David Schuller 
mailto:dj...@cornell.edu>> wrote:

I have some SIRAS data of a known structure. I want to get the
isomorphous and anomalous occupancy and phasing power from my data.
What's the best software to do this?

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] A question on protein microheterogenity for crystalization

[Bosch, Juergen]

Hi Acoot,

since they behave differently on IEX, they are different - you would introduce 
heterogeneity into your crystallization setup, which usually is not a good idea 
for crystallization in general.
Are you using Benzonucleases by any chance in your preparation and if not, that 
may explain the different populations of your protein. Your protein may bind 
some short DNA chunks carried over during your lysis process leading to 
differently charged species on the IEX column. On an SDS gel these complexes 
are separated and your protein runs at an identical molecular weight. An 
alternative explanation would be multiple folded states that may be at 
equilibrium and under certain conditions e.g. pH, salt concentration may be 
pushed in one predominant species.
Also is several 2 or 10 peaks ? If you have e.g. an N-terminal His6-tag you 
might end up with truncation products and say your protein is rather large e.g. 
80 kDa you might not be able to see the molecular weight difference either by 
SEC or SDS-PAGE if you are running a low percentage gel. A protein of 72 kDa 
might look like 80 kDa but you most likely will have charge differences 
distinguishable in IEX. If you have a His6-tag, run a Western on it to identify 
single or multiple bands.

Good luck,

Jürgen

On Dec 14, 2013, at 7:16 AM, Acoot Brett 
mailto:acootbr...@yahoo.com>> wrote:

Dear All,

When I purified my protein by ion exchange chromatography for crystallization, 
there were several peaks containing the target protein as analyzed by SDS-PAGE. 
All these peaks have the same MW as determined by gel filtration coupled MALLS.

For crystallization purpose, can I merge the corresponding ion exchange 
chromatography peaks together? Otherwise the protein yield will be too low. And 
how to explain the heterogeneity by ion exchange chromatography in this 
situation?

I am looking forward  to getting a reply from you.

Acoot

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Wrong Space Group?

[Bosch, Juergen]

2.8 is not a terrible resolution to try out Buccaneer or Parrot. Your terrible 
R-factor might be due to a shift in residues perhaps ? Your After-building map 
does not show much of a side chain density to judge if you are in frame or off.
But the elongated helix is in my eyes convincing enough.

One thing you might want to try is to use composite omit maps to reduce your 
bias from MR and verify that what you built is indeed correct.

Jürgen

On Dec 13, 2013, at 2:44 PM, D Bonsor 
mailto:dbon...@ihv.umaryland.edu>> wrote:

Dear all,

I have collected ~160 degrees of data on a new crystal form of a protein which 
has already been solved. Data was processed with XDS and reindex, scaled and 
truncated with Aimless. Both XDS and Pointless suggested a Laue group of  
P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall 
Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a 
completeness of 99.1% and resolution of 2.8Ang.

With cell dimensions of 63.1 63.1 243, only one protein chain can be found in 
the asymmetric unit (two copies would leave a solvent content of 8%). I ran 
phaser with all alternative space groups and a single solution in P6522 with a 
TFZ of 10.0.

I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I 
open the structure and map in Coot and could see that there was a large 
conformational change of helix-turn-helix actually becoming just a long helix 
(https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then 
dimerizing through the long helix with one of the symmetry mates.

This section was rebuilt 
(https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through 
Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of 
the structure I see nothing else really to be modeled. Nothing that could bring 
the Rfactors down to a reasonable range.

I have therefore tried several things. I ran the structure through Zanuda 
server to look at other space group possibilities. The server suggested I was 
in the correct space group. However I did reprocess the data to P6, P3, P312, 
P321, C2221, P2 and C2, and reran phaser in "search all alternative space 
groups" using the original search model but found no solutions. I did reprocess 
the data in P1, though I did not collect enough data.

Twinning tests show no twinning. Although that does not mean there is no 
twinning, I can see that P6522 has no twin laws. Does that mean no twinning can 
occur in P6522 or that it can occur but there is no law to be able to separate 
the amplitudes?

I also collected data on a single point mutation of the protein. Although this 
diffracted to a slightly weaker resolution (3.2Ang), I also observe the same 
problem of good maps in P6522 but no solution in the groups described above, a 
clear indication that this helix has elongated but terrible Rfactors.

Based upon that the maps look good in P6522 do you believe that I have solved 
the structure in the correct space group but my data collection is at fault or 
in fact that I have some form of pseudosymmetry or something else going on and 
that the space group has lower symmetry but not in the space groups I have 
checked. Or is it something else.

Any suggestions, criticisms or you need further information please contact me 
and enjoy your weekend.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Off topic: Problem in reproducing crystal screen

[Bosch, Juergen]

another option might be the age of the screen perhaps ?
very silly suggestion/question: your pH meter is calibrated before you make 
your measurements right ?

If you have a nano-pH probe (anything that can be added to a 96well reservoir 
well) you could test what the final pH of your composition is in which you do 
obtain crystals and then pH the final mixture to that value with the same 
electrode.

Jürgen

On Dec 9, 2013, at 4:59 AM, Evgeny Osipov 
mailto:e.m.osi...@gmail.com>> wrote:

Hello, Nasir,
What concentration of your ammonium sulphate stock solution? Do your
solution remains immiscible without ammonium sulphate?May be addition of
concentrated solution of ammonium sulphate to PEG MME could cause phase
separation? Anyway, try to heat your solution a bit as recommended by
Tim or use ammonium sulphate stock solution with low ( probably 1 to 2
M) concetration and tell us if it works
06.12.2013 20:39, Tim Gruene пишет:
Dear Nazia Nasir,

did you warm up the solution a little? This makes working with PEG
solutions a lot easier. You may also note the Hampton do not maintain
the pH: they use the buffers as indicated but this does not mean that
the final solution still has pH 6.5. With your ingredients it probably
is, but e.g. with high concentrations of imidazole the final pH would
differ a lot.

Best,
Tim

On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote:
I have obtained crystals in Hampton Crustal Screen 2, condition 26.The
condition has the following components:
1. 0.2 M ammonium sulphate
2. 0.1 M MES buffer pH 6.5
3. 30% PEG mononmethyl ether 5,000.

When I try to prepare the screen in-house, I use the Hampton stocks of 3.5
M ammonium sulphate and 50%  PEG mononmethyl ether 5,000. I make the MES
buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH
accurately. The water I use is filtered MilliQ. However, the PEG MME and
the rest of the solution remains immiscible and thus, forms droplets of
separated PEG when I set up crystallization. The original condition from
Hampton has no such problems and gives good crystals.

Can anyone let me know how to overcome the problem insolubility of PEG MME
in the buffer mix? I have not faced such problems with other conditions
which I prepare in house and have PEG in them. Further, it's not possible
for me to use the original screen all the time.

Thanks a lot!


Regards



--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?

[Bosch, Juergen]

What is the solution to this?
Hi Meisam,
you have it, it is just three molecules in the asu. Look at the overall crystal 
lattice packing and see if you have contacts supporting each molecule. Generate 
a large representation of your symmetry mates, I suspect you have a channel in 
your crystal lattice, explaining why your Matthews coefficient leads you in a 
wrong direction.
If you are not convinced, calculate a selfrotation function with Molrep and 
look at the Chi=120 section and compare it to the Chi=90.

At least judging from your R-factors, if I understand you correctly you have 
not done much of a refinement yet. Have you performed a real space refinement 
and adjusted side chains etc. ?

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Nov 21, 2013, at 12:35 PM, Meisam 
mailto:meisam.nosr...@gmail.com>> wrote:

Dear CCP4ers

I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% 
linear Rfactor.
The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric 
unit, 53% for 4 molecules, and 36% for 5 molecules.
Molecular replacement just finds 3 molecules in the asymmetric unit. Running 
Phaser also gives a partial solution with 3 molecules.
When I refine just the back bone of the protein for the 3 molecules the 
Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular 
replacement on the refined structure again, and I fix it as a model to search 
for another molecule, it still does not find it.

I have attached a photo to show the density for the fourth molecule in the 
asymmetric unit.

What is the solution to this?

Thanks in advance for your help

Meisam

Re: [ccp4bb] PDB structure validation

[Bosch, Juergen]

You do use Coot and look at the density plots right ?
Phenix will essentially do the same but in text form (unless you use the GUI). 
You can also do this with Solve/Resolve and you can write your own script with 
CCP4 available tools to do the same RSR fit diagram.
Jürgen

On Oct 31, 2013, at 11:43 AM, Debasish Chattopadhyay wrote:


Yes, there remains many questions beyond Adit.
I do want to emphasize that the new scrutiny in PDB is very good since it now 
includes a density fitting analysis (everything in the structure should be the 
density, right) etc.   But one has to go through the submission to generate the 
report and then has to resubmit the coordinates again if corrections are 
necessary.
We know about Molprobity, structures with good Molprobity score and clash score 
can still have some issues.
I just saw the note from Pavel and I need to check the option in Phenix.

Thanks all.

Debasish

From: longingforadmiss...@gmail.com 
[mailto:longingforadmiss...@gmail.com] On Behalf Of Mahesh Lingaraju
Sent: Thursday, October 31, 2013 10:32 AM
To: Debasish Chattopadhyay
Subject: Re: [ccp4bb] PDB structure validation

Hi

One can do an unofficial validation in Adit server ( one of the pdb deposition 
services) but i have found that although it is almost the same thing, it does 
not provide a lot of information that we get from the official report.

I found that the apps from the PDB_redo help a lot in ironing out the kinks at 
the final stages of structure refinement that are otherwise not so obvious.

Thanks

Mahesh



On Thu, Oct 31, 2013 at 11:25 AM, Debasish Chattopadhyay 
mailto:debas...@uab.edu>> wrote:
I was wondering if there is a way to generate a PDB validation report before 
depositing the coordinates so that one can go back and make necessary 
corrections to the file before deposition.  It will save a lot time and perhaps 
would improve the quality of deposited structures.

Debasish Chattopadhyay

University of Alabama at Birmingham
CBSE-250
1025 18th Street South, Birmingham, Al-35294
USA
Ph: (205)934-0124; Fax: 
(205)934-0480


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] PDB structure validation

[Bosch, Juergen]

yes, indeed.
visit this site and make sure you get all green lights
http://molprobity.biochem.duke.edu

Jürgen

On Oct 31, 2013, at 11:25 AM, Debasish Chattopadhyay wrote:

I was wondering if there is a way to generate a PDB validation report before 
depositing the coordinates so that one can go back and make necessary 
corrections to the file before deposition.  It will save a lot time and perhaps 
would improve the quality of deposited structures.

Debasish Chattopadhyay

University of Alabama at Birmingham
CBSE-250
1025 18th Street South, Birmingham, Al-35294
USA
Ph: (205)934-0124; Fax: (205)934-0480


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Unrelated - Emulsiflex C3 leaking

[Bosch, Juergen]

We've had that leak as well some time ago. Dismantle and reassemble it is the 
trick. Just a slight angle when tightening it will lead to that problem. This 
is usually the case because of overtughtening the reservoir to the main part of 
the machine. Keep it always straight when closing it again.
Jürgen 

Sent from my iPad

> On Oct 29, 2013, at 11:44, "Chiara Rapisarda"  
> wrote:
> 
> Hi all,
> 
> sorry for the unrelated e-mail, but we have an emulsiflex C3 and it leaks 
> from the chamber that contains the sample as I show in the figure. The 
> Avestin European support was not very helpful, has anybody experienced a 
> similar leak and has succeeded in fixing it?
> 
> Chiara
> 
> 

Re: [ccp4bb] Biacore/SPR

[Bosch, Juergen]

BiaCore 3000 or if you can afford the T200.
Proteon XPR36 would be a cheaper Option and should work as well.
Jürgen

On Oct 28, 2013, at 10:54 AM, Gang Dong wrote:

We mainly measure protein-protein interactions (sometimes protein-small 
molecules). Thanks! _Gang

From: Bosch, Juergen [mailto:jubo...@jhsph.edu]
Sent: Monday, October 28, 2013 3:17 PM
To: Gang Dong
Cc: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Biacore/SPR

Protein-protein interaction?
protein-small molecule interactions ?
Epitope mapping of mABs ?
Could you specify what you would like to do, as different models are good for 
different things.
Jürgen

On Oct 28, 2013, at 10:08 AM, Gang Dong wrote:


Dear all,

Could anyone tell me your experience with Biacore (any models) or a similar 
SPR-based technology for measuring interaction kinetics? I also want to know 
the price ranges to discuss with our department/facility managers.

Thanks,
Gang



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Biacore/SPR

[Bosch, Juergen]

Protein-protein interaction?
protein-small molecule interactions ?
Epitope mapping of mABs ?
Could you specify what you would like to do, as different models are good for 
different things.
Jürgen

On Oct 28, 2013, at 10:08 AM, Gang Dong wrote:

Dear all,

Could anyone tell me your experience with Biacore (any models) or a similar 
SPR-based technology for measuring interaction kinetics? I also want to know 
the price ranges to discuss with our department/facility managers.

Thanks,
Gang



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] a problem when using NOMACHINE Player from OSX 10.7.4

[Bosch, Juergen]

check out the hidden .nx folder in your home directory.
You should have a folder named config.
You may have a .nxs file or .cfg file there just edit the file with a text 
editor.
You probably need to change this value:


If you have a retina MacBook I guess you need to double that value perhaps.

Jürgen

On Sep 18, 2013, at 10:06 PM, Alexander Aleshin wrote:

> I've got a question to people using a remote access to synchrotron computers. 
> I've got Macbook pro with Mac OSX 10.7.4, which requires the use of nxplayer 
> instead of commonly used nxclient.  It works, but the size of emulated screen 
> gets twice bigger than the size of my computer's screen. As a result I can 
> see only a half of it. Does anybody know how to fix this problem?
> 
> Alexander Aleshin

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Adding ligand to crystallization drop

[Bosch, Juergen]

But you have not tested in the beam if the visually happy crystals diffract 
right ?
I would do this first before consuming your precious ligands in worthless drops.
Jürgen

On Sep 12, 2013, at 10:34 AM, Christopher Browning wrote:

 Right now I've tried 5, 10 and 16% DMSO (due to variations in the final ligand 
conc.) and the crystals visually look happy.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] over-expression strategies

[Bosch, Juergen]

Is your gene of interest smaller than 750bp ?
Then I would synthesize the gene with IDT and optimize it for E.coli that's 139$
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://lupo.jhsph.edu

On Sep 12, 2013, at 10:23, "Elias Fernandez" 
mailto:efern...@utk.edu>> wrote:

Dear CCP4ers,
We’ve been struggling with little (nearly none) expression of our protein, in 
both E coli and with in vitro transcription/translation methods. It appears 
that our mRNA has high 2’ structure with a low dG (theoretically ~760kcal/mol). 
If this is indeed the source of our problem, are there any potential strategies 
to either disrupt the mRNA structure chemically (in E coli or in vitro) or with 
 thermophile expression systems for expression at higher temperatures?
Regards,
Elias

Re: [ccp4bb] Adding ligand to crystallization drop

[Bosch, Juergen]

Have a look at these two papers:
http://www.ncbi.nlm.nih.gov/pubmed/17004709
http://www.ncbi.nlm.nih.gov/pubmed/19929835

When you say high DMSO, how much is that in % ? Do you know if your crystals 
survive that much percentage DMSO even without ligand ?

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Sep 12, 2013, at 6:25 AM, Christopher Browning wrote:

Hi Again,

Thanks for the suggestions. I just tried it at 2 ligand concentrations and the 
crystals seem not to mind. The drop does go a little cloudy but I can't say if 
this is protein precipitation, or the ligand coming out of solution. My feeling 
its a bit of protein because of the high DMSO conc. being added. I'll 
co-crystallize just to have a backup!

Cheers

C



--
Christopher Browning, Ph. D

Vertex Pharmaceuticals (Europe) Ltd
86-88 Jubilee Avenue
Milton Park
Abingdon
Oxfordshire
OX14 4RW
United Kingdom

Tel +44 (0) 1235 438327
christopher_b_brown...@vrtx.com
www.vrtx.com

From: , "Nicholas [E] (NIH/NIDDK)" 
mailto:noin...@niddk.nih.gov>>
Date: Thursday, 12 September 2013 10:21
To: Christopher Browning 
mailto:christopher_b_brown...@vrtx.com>>, 
"CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: RE: Adding ligand to crystallization drop

Chris,

Bottom line is that it is all dependent on how your ligand interacts with your 
protein, if there are any conformational changes, and how your crystals behave 
in such a dilution and high concentration of ligand.  You just have to test it 
and see, no way to know for sure until you do the experiments and see.  
However, there are some things to look for along the way.  For example, do your 
crystals crack after introducing the ligand or do they start to dissolve and 
lose their nice edges, or turn opaque?  you will probably also want to do a 
buffer only control without ligand to ensure that the crystals are ok with the 
dilution and change of environment.

I assume the native crystals diffract well enough to solve the structure.  You 
would want to test crystals before any soaking and then with buffer only and 
then with ligand, to ensure diffraction is retained to a level useful enough 
for structure determination.  I did quite a bit of crystal soaking in grad 
school and it is something you just have to try since every crystal/protein is 
different. my experience is that soaking can often lead to a slight loss of 
resolution but typically you still get the information you are seeking if the 
crystals survive.

with that being said, soaking can sometimes lead to partial occupancy of your 
ligand.  So if you are able to do co-xtallization, i think that would be 
preferred and doesn't seem like too much additional effort assuming you are 
getting crystals in the same conditions as with native protein only.  and with 
co-xtallization, I often see slightly better resolution that with native 
protein only (not always the same condition though unfortunately).  But again, 
no way to know how your particular protein/crystal will behave without doing 
some pilot experiments first.

Good luck!





Cheers,
Nick





[ Nicholas Noinaj ]
the Buchanan Lab
Laboratory of Molecular Biology
LMB-NIDDK, NIH
50 South Drive, Room 4505
Bethesda, MD  20892-8030
1-301-594-9230 (lab)
1-859-893-4789 (cell)
noin...@niddk.nih.gov

[ the Buchanan Lab ]
http://www-mslmb.niddk.nih.gov/buchanan/













From: Christopher Browning [mailto:christopher_b_brown...@vrtx.com]
Sent: Thursday, September 12, 2013 4:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Adding ligand to crystallization drop

Hi,

I've just got a quick question about getting ligands bound to proteins in 
crystals. I've managed to co-crystallize my proteins with the various ligands, 
and I'm aware that soaking will work as well but I want to speed up the process.

Would this work? If I had a bunch of crystals that have grown in their drop, 
can I pipette the ligand (say 0.6ul to get a desired conc.) onto the drop and 
just let the crystals soak that way. Any dilution that would have occurred when 
then readjust as the drop will be sealed again. Perhaps the crystals would get 
a little stressed when adding the high concentrated ligand and this method 
would not work…?

Thanks,

Chris



--
Christopher Browning, Ph. D

Vertex Pharmaceuticals (Europe) Ltd
86-88 Jubilee Avenue
Milton Park
Abingdon
Oxfordshire
OX14 4RW
United Kingdom

Tel +44 (0) 1235 438327
christopher_b_brown...@vrtx.com
www.vrtx.com
This email message and any attachments are confidential 

Re: [ccp4bb] Quick resolution cutoff survey

[Bosch, Juergen]

Dear CCP4bb,

almost 12h have passed since I posted this question to the board. Since some of 
us get daily or weekly digests I will hold off with revealing the results. But 
the replies thus far are really interesting and exciting, and this is without 
any sarcasm tags. However we have only sampled 1.5% of the CCP4 community thus 
far.

I will try to have an almost complete reply compiled to all questions raised in 
the comments box, say in about one week.

Thanks to all those that participated,

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Aug 28, 2013, at 11:32 AM, Bosch, Juergen wrote:

Since we keep discussing resolution cutoffs and the benefits of not to include 
all data etc.
I thought I would crowd source your opinion on this particular data set.

processed with XDS, here's the XSCALE.LP output:

 SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
 RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR 
COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
   LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected
  Corr

 9.4353651009  1028   98.2%   1.7%  2.1% 
5351   68.92 1.8%   100.0* 20.698 691
 6.67   101531756  1760   99.8%   2.2%  2.4%
10134   58.30 2.4%   100.0*   -120.6741404
 5.44   131142217  2223   99.7%   3.4%  3.5%
13097   42.38 3.7%99.9*   -100.7161845
 4.71   152732583  2592   99.7%   3.2%  3.1%
15259   46.24 3.5%99.9*   -140.7332212
 4.22   171832907  2934   99.1%   3.2%  3.2%
17173   45.14 3.6%99.9*   -160.7222538
 3.85   190103183  3217   98.9%   4.4%  4.1%
19000   37.38 4.8%99.9*   -160.7172794
 3.56   207643441  3473   99.1%   5.9%  5.6%
20752   30.36 6.5%99.9*   -130.7543061
 3.33   225163681  3712   99.2%   8.8%  8.5%
22507   22.60 9.7%99.7*   -110.7373293
 3.14   247353963  4001   99.1%  12.4% 13.0%
24725   16.7713.5%99.5*-80.6963565
 2.98   259314127  4161   99.2%  17.2% 18.1%
25924   12.8218.7%99.2*-60.7103751
 2.84   265214291  4386   97.8%  25.4% 26.9%
264959.2627.6%98.3*-40.6833809
 2.72   203573495  4592   76.1%  27.6% 29.3%
202777.9030.2%97.9* 00.7062826
 2.61   159172860  4768   60.0%  33.7% 35.0%
158396.4137.0%96.6*-20.6972171
 2.52   129492394  4944   48.4%  42.5% 45.1%
128774.9146.8%95.3* 00.6941692
 2.43   103101993  5097   39.1%  47.8% 50.7%
102304.0853.0%94.4*-20.6701295
 2.3681801693  5309   31.9%  56.4% 60.1% 
80793.1263.0%92.2*-20.671 961
 2.2960751381  5441   25.4%  69.9% 72.5% 
59712.2878.9%87.2*   -100.618 643
 2.2240011077  5610   19.2%  82.9% 81.9% 
38931.7896.0%80.7*-80.633 340
 2.162491 799  5771   13.8%  78.0% 83.6% 
23761.4792.9%75.9*-40.586 154
 2.11 786 367  59016.2% 103.0%106.4%  
6660.87   129.9%63.1*120.580  28
total  281631   49217 80920   60.8%   7.1%  7.2%   
280625   21.54 7.8%99.9*-70.706   39073

And here's the link so you can voice your opinion in a Survey Monkey. Results 
from this survey will be reported back to the CCP4bb.

http://www.surveymonkey.com/s/YNDKM6G

Thanks for your participation and no there's no iPad or iPod-touch to win, and 
you also don't have to disclose your email.

The survey has only two questions, one is just a click the other one you 
provide your opinion on your decision.

Thanks,

Jürgen

P.S low resolution shell starts at 44 Å - 9.43
P.P.S. the Table1 will be revealed once I report back the outcome of this 
survey.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 Nort

[ccp4bb] Quick resolution cutoff survey

[Bosch, Juergen]

Since we keep discussing resolution cutoffs and the benefits of not to include 
all data etc.
I thought I would crowd source your opinion on this particular data set.

processed with XDS, here's the XSCALE.LP output:

 SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
 RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR 
COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
   LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected
  Corr

 9.4353651009  1028   98.2%   1.7%  2.1% 
5351   68.92 1.8%   100.0* 20.698 691
 6.67   101531756  1760   99.8%   2.2%  2.4%
10134   58.30 2.4%   100.0*   -120.6741404
 5.44   131142217  2223   99.7%   3.4%  3.5%
13097   42.38 3.7%99.9*   -100.7161845
 4.71   152732583  2592   99.7%   3.2%  3.1%
15259   46.24 3.5%99.9*   -140.7332212
 4.22   171832907  2934   99.1%   3.2%  3.2%
17173   45.14 3.6%99.9*   -160.7222538
 3.85   190103183  3217   98.9%   4.4%  4.1%
19000   37.38 4.8%99.9*   -160.7172794
 3.56   207643441  3473   99.1%   5.9%  5.6%
20752   30.36 6.5%99.9*   -130.7543061
 3.33   225163681  3712   99.2%   8.8%  8.5%
22507   22.60 9.7%99.7*   -110.7373293
 3.14   247353963  4001   99.1%  12.4% 13.0%
24725   16.7713.5%99.5*-80.6963565
 2.98   259314127  4161   99.2%  17.2% 18.1%
25924   12.8218.7%99.2*-60.7103751
 2.84   265214291  4386   97.8%  25.4% 26.9%
264959.2627.6%98.3*-40.6833809
 2.72   203573495  4592   76.1%  27.6% 29.3%
202777.9030.2%97.9* 00.7062826
 2.61   159172860  4768   60.0%  33.7% 35.0%
158396.4137.0%96.6*-20.6972171
 2.52   129492394  4944   48.4%  42.5% 45.1%
128774.9146.8%95.3* 00.6941692
 2.43   103101993  5097   39.1%  47.8% 50.7%
102304.0853.0%94.4*-20.6701295
 2.3681801693  5309   31.9%  56.4% 60.1% 
80793.1263.0%92.2*-20.671 961
 2.2960751381  5441   25.4%  69.9% 72.5% 
59712.2878.9%87.2*   -100.618 643
 2.2240011077  5610   19.2%  82.9% 81.9% 
38931.7896.0%80.7*-80.633 340
 2.162491 799  5771   13.8%  78.0% 83.6% 
23761.4792.9%75.9*-40.586 154
 2.11 786 367  59016.2% 103.0%106.4%  
6660.87   129.9%63.1*120.580  28
total  281631   49217 80920   60.8%   7.1%  7.2%   
280625   21.54 7.8%99.9*-70.706   39073

And here's the link so you can voice your opinion in a Survey Monkey. Results 
from this survey will be reported back to the CCP4bb.

http://www.surveymonkey.com/s/YNDKM6G

Thanks for your participation and no there's no iPad or iPod-touch to win, and 
you also don't have to disclose your email.

The survey has only two questions, one is just a click the other one you 
provide your opinion on your decision.

Thanks,

Jürgen

P.S low resolution shell starts at 44 Å - 9.43
P.P.S. the Table1 will be revealed once I report back the outcome of this 
survey.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Resolution, R factors and data quality

[Bosch, Juergen]

What a statement !
Give reviewers maps, I agree however, what if the reviewer has no clue of these 
things we call structures ? I think for those people table 1 might still 
provide some justification. I would argue it should go into the supplement at 
least.

Jürgen 

Sent from my iPad

On Aug 28, 2013, at 5:58, "Bernhard Rupp"  wrote:

>> We don't currently have a really good measure of that point where adding
> the extra shell of data adds "significant" information 
>> so it probably isn't something to agonise over too much. K & D's paired
> refinement may be useful though.
> 
> That seems to be a correct assessment of the situation and a forceful
> argument to eliminate the
> review nonsense of nitpicking on  values, associated R-merges, and
> other
> pseudo-statistics once and for good. We can now, thanks to data deposition,
> at any time generate or download the maps and the models 
> and judge for ourselves even minute details of local model quality from
> there. 
> As far as use and interpretation goes, when the model meets the map is where
> the rubber meets the road.
> I therefore make the heretic statement that the entire table 1 of data
> collection statistics, justifiable in pre-deposition times 
> as some means to guess structure quality can go the way of X-ray film and be
> almost always eliminated from papers. 
> There is nothing really useful in Table 1, and all its data items and more
> are in the PDB header anyhow. 
> Availability of maps for review and for users is the key point.
> 
> Cheers, BR

Re: [ccp4bb] Resolution, R factors and data quality

[Bosch, Juergen]

Hi Jim,

all data is good data - the more data you have the better (that's what they say 
anyhow)

Not everybody is adopting to the Karplus Diederich paper as quickly as you do. 
And not to be confused with the Diederichs and Karplus paper :-)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689524/
http://www.ncbi.nlm.nih.gov/pubmed/22628654

My models get better by including the data I had been omitting before, that's 
all that counts for me.

Jürgen

P.S. reminds me somehow of those guys collecting more and more data - PRISM 
greetings

On Aug 27, 2013, at 8:29 PM, Jim Pflugrath wrote:

I have to ask flamingly: So what about CC1/2 and CC*?

Did we not replace an arbitrary resolution cut-off based on a value of Rmerge 
with an arbitrary resolution cut-off based on a value of Rmeas already?  And 
now we are going to replace that with an arbitrary resolution cut-off based on 
a value of CC* or is it CC1/2?

I am asked often:  What value of CC1/2 should I cut my resolution at?  What 
should I tell my students?  I've got a course coming up and I am sure they will 
ask me again.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] 
on behalf of Arka Chakraborty 
[arko.chakrabort...@gmail.com]
Sent: Tuesday, August 27, 2013 7:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Resolution, R factors and data quality

Hi all,
does this not again bring up the still prevailing adherence to R factors and 
not  a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans 
has indicated).?
The way we look at data quality ( by "we" I mean the end users ) needs to be 
altered, I guess.

best,

Arka Chakraborty

On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans 
mailto:p...@mrc-lmb.cam.ac.uk>> wrote:
The question you should ask yourself is "why would omitting data improve my 
model?"

Phil

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?

[Bosch, Juergen]

Hi Mahesh,

if you use Refmac, then you can tell it to refine the twin fraction, no need to 
tell it the twin law as Refmac will figure it out. If you use phenix, you 
explicitly tell it the twin law and refine then with it. You can get the 
possible twin laws by running phenix.xtriage and looking at the log file.

For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr, even 
though your Rfactors are high. If that is the case then you are likely correct 
with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you did add 
some waters to your structure already right ? if not the go water picking via 
Coot.

Have you been converted to XDS now ? Welcome to the club.

Jürgen

On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote:

Hello everyone,

I collected a dataset which looked like it is twinned ( or a really long axis 
in the cell)  and did not process in HKL2000 and MOSFLM but with some of help 
and suggestions from CCP4BB, XDS was able to process it. The data looks good 
upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost 
complete. I am beginning to wonder if the data is really twinned as it has the 
characteristics of non- merohedral twinning:
In the images some of the reflections are sharp while some are split and one of 
the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 
90 90 90)

is there anyway to work around this ? or collecting better data is the only 
solution ?

Any help is deeply appreciated

Thanks

Mahesh

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] cryoprotection

[Bosch, Juergen]

increase your PEG3350 >27% and keep the other components at their current 
concentration. You can also add glycerol or ethylene glycol into the mix.

If you have multiple crystals then try various variants.

Jürgen

On Aug 23, 2013, at 1:52 PM, Uday Kumar wrote:

Hello

Can anyone suggest a cryoprotectant for the following crystallization condition

0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Low 280 absorbance imidazole?

[Bosch, Juergen]

But when it sits and crystallizes it is cleaner - unless some opportunistic 
fungal contamination helps you trim off those nasty loops that you would have 
omitted anyhow from your model.

Jürgen

P.S. I like kangaroo's in particular when served with red wine and medium rare 
next to some garlic mashed potatoes

On Aug 22, 2013, at 2:17 PM, Ed Pozharski wrote:

On Thu, 2013-08-22 at 13:58 -0400, Bosch, Juergen wrote:
well if we hit the timer after lysis, say via cell disruptor then I
have my eluted protein in less than 1 hour, including 40 minutes batch
binding.

then proceeds to wait six weeks for crystals to appear... :)

Cheers,

Ed.

PS.  There are many ways to skin a kangaroo and ultimately indeed all
things serve the Beam.

--
"I'd jump in myself, if I weren't so good at whistling."
  Julian, King of Lemurs


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Low 280 absorbance imidazole?

[Bosch, Juergen]

Hi Ed,

well if we hit the timer after lysis, say via cell disruptor then I have my 
eluted protein in less than 1 hour, including 40 minutes batch binding. If I'm 
pressed for time I bind only 20 minutes and then can have the protein ready for 
the next step within 30 minutes after lysis. Some of you are then still 
centrifuging and have not even started to load the column. [Quick jump to last 
paragraph for the reason]

I see the disadvantage more in occupying an Akta for something that could be 
easily done without one. Also a pump with gradient mixer may be an alternative.
But every protein is different and you should obey to your protein and not 
stick to what you think is best, trial and error and hopefully moving fast to 
some conclusions on how to best purify your protein.

What I noticed over the years is that frequently if the initial capturing step 
takes too long you will end up with protein that is not necessarily as 
functional as you wanted it. You can add cOmplete tablets (if you don't work on 
proteases) of course, but sometimes you can't do that and then it gets 
important to clean up the sample ASAP.

I had my protein once almost completely disappear because I thought I could go 
quickly shopping while the centrifuge is spinning down the lysate. So I added 
an extra hour instead of 30 minutes. Well that purification ended in nothing. I 
believe I learnt something - did I ?

Just a thought

Jürgen


On Aug 22, 2013, at 1:47 PM, Ed Pozharski wrote:

On Thu, 2013-08-22 at 10:07 -0400, Bosch, Juergen wrote:
Yes, I'm also surprised why people run gradients for the capturing
step ?

Because we can.  Joking aside, I've seen some examples where protein
eluted at relatively low imidazole and upon running the gradient there
remains some (minimal) overlap with non-specific binders.  Mainly I just
never seen consensus on what is the right imidazole concentration for
the wash buffer (10mM? 50mM? may even depend on expession
circumstances), running the gradient takes that uncertainty away.  Also,
batch method probably leaves more contamination behind.

One can run imidazole gradient in 1-2 hours, batch is not really much
faster, if at all.  So if one has FPLC access, doing imidazole gradient
seems like a good standard policy.  Benefits might be minor and rare,
but it is not much more work and certainly not worse than batch in any
respect.

My two cents (which in Russia turns into three rusty kopecks),

Ed.

--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
   Julian, King of Lemurs


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Low 280 absorbance imidazole?

[Bosch, Juergen]

Yes, I'm also surprised why people run gradients for the capturing step ? How 
often is your desired protein spread out throughout the whole gradient ? So 
what's the point the of the gradient if in the end you merge the whole peak 
again ?
Maybe for a pilot study to identify how much imidazole could I add to my 
binding buffer I can see the point of the gradient, but otherwise batch binding 
and step elution is just fine (and faster).

We do batch binding in the presence of some imidazole 10 -25 mM then do a step 
elution with 250 - 500 (depending on His6 or His12) if we use imidazole for 
elution. Alternatively we do low-pH elution e.g. if your protein requires a 
divalent ion or elution with EDTA at a more physiological pH if no divalent ion 
is required by our protein.

The cleanup is a Resource Q or Resource S followed by size exclusion or other 
steps in between. If the captured protein is not >50% in the first step then we 
try to enrich e.g. by HIC or simply reducing the amount of resin used in the 
first capturing step. My lab is a big fan of Talon Celltru lately. And yes, 
they really should send me some free samples for making this advertisement :-)

Jürgen

On Aug 22, 2013, at 9:37 AM, David Mueller wrote:

I've never done the experiment, but it is easy to determine the absorption 
spectra  of imidazole at different pH values and see if there is an isosbestic 
point - and then plot  (A280 minus the Abs at the isobestic point),  vs pH, and 
see if the pKa is about 7.0.   Imidazole is aromatic as the free base but not 
as the protonated species, so I expect a change in the UV spectrum vs pH.   
Qiagen used to recommend not doing a gradient of imidazole to elute the protein 
but simply stepping the protein off with a high concentration of imidazole - it 
works for me.


On Thu, Aug 22, 2013 at 8:03 AM, Gloria Borgstahl 
mailto:gborgst...@gmail.com>> wrote:
The absorbance is due to imidazole, for sure.
We do a lot of this type of purification in my lab and we
have definitely had some low abundance proteins hide under the imidazole 
gradient.
Here is how we deal with it.
We expect the proteins to elute around 100-350 mM imidazole, so we run SDS-PAGE 
on every other fraction in that area
to detect the protein.
We do 3-5 clarified lysate loads and low imidazole washes to increase the 
amount of our desired protein on the column before
we start the imidazole gradient.
Always have a few CV of baseline absorbance before you start the gradient.
That's all I know, G


On Wed, Aug 21, 2013 at 10:45 AM, Ed Pozharski 
mailto:epozh...@umaryland.edu>> wrote:
According to Qiagen


> Since imidazole absorbs UV radiation at 280 nm, an elution profile
> measured at 280 nm while purifying a 6xHis tagged protein by FPLC will
> show an increase in absorbance above the background signal allowing
> quantitation of your protein. The absorbance of imidazole can vary
> depending on its source and purity, but elution buffer containing 250
> mM imidazole usually has an A280 of 0.2–0.4.

Cheers,
Ed.


On Wed, 2013-08-21 at 11:25 -0400, Edward A. Berry wrote:
> Would you happen to know what the A280 of a 1M (or .1 M) solution is?
> I wonder if this absorbance is really due to impurities or is the tail of a 
> weak absorbance band of imidazole itself. I
> notice A280 is not one of the properties sigma lists for its imidazole.
>
> Jan wrote:
> > Hi Bernhard,
> > this one works for us:
> > Sigma BioUltra imidazole, >99.5% by GC, 56749
> >
> > Cheers,
> > Jan
> > --
> > Jan Abendroth
> > Emerald BioStructures
> > Seattle / Bainbridge Island WA, USA
> > home: Jan.Abendroth_at_gmail.com
> > work: JAbendroth_at_embios.com
> > http://www.emeraldbiostructures.com
> >
> > On Aug 21, 2013, at 7:33 AM, Bernhard Rupp 
> > mailto:hofkristall...@gmail.com> 
> > >> wrote:
> >
> >> Hi Fellows,
> >> could someone please point me towards the source of a known high purity 
> >> imidazole
> >> with low absorbance at 280 nm? I am facing the problem of detecting a low 
> >> absorption protein
> >> in high imidazole background after IMAC gradient elution. In the UV 
> >> spectra of the
> >> 2 imidazoles I checked there is some contaminant that absorbs at 280…
> >> Thx, BR
> >> 
> >> Bernhard Rupp
> >> Marie Curie Incoming International Fellow
> >> Innsbruck Medical University
> >> Schöpfstrasse 41
> >> A 6020 Innsbruck – Austria
> >> +43 (676) 571-0536
> >> bernhard.r...@i-med.ac.at 
> >> >
> >> 
> >> Dept. of Forensic Crystallography
> >> k.-k. Hofkristallamt
> >> Vista, CA 92084
> >> 001 (925) 209-7429
> >> b...@ruppweb.org

Re: [ccp4bb] Low 280 absorbance imidazole?

[Bosch, Juergen]

How about low pH elution or EDTA as alternative ?
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://lupo.jhsph.edu

On Aug 21, 2013, at 14:40, "Prof. Dr. Arne Skerra" 
mailto:ske...@tum.de>> wrote:

Hi Bernhard,

We know this problem since the very early days of the His-tag and have been 
using high purity imidazole from Merck since then with success, which 
essentially shows no absorption at 280 nm. If you like I can enquire the 
current order number in my lab.

Cheers, ASk


Am 21.08.2013 um 16:33 schrieb Bernhard Rupp:

Hi Fellows,

could someone please point me towards the source of a known high purity 
imidazole
with low absorbance at 280 nm? I am facing the problem of detecting a low 
absorption protein
in high imidazole background after IMAC gradient elution. In the UV spectra of 
the
2 imidazoles I checked there is some contaminant that absorbs at 280…

Thx, BR


Bernhard Rupp
Marie Curie Incoming International Fellow
Innsbruck Medical University
Schöpfstrasse 41
A 6020 Innsbruck – Austria
+43 (676) 571-0536
bernhard.r...@i-med.ac.at

Dept. of Forensic Crystallography
k.-k. Hofkristallamt
Vista, CA 92084
001 (925) 209-7429
b...@ruppweb.org
b...@hofkristallamt.org
http://www.ruppweb.org/
---


Prof. Dr. Arne Skerra
Lehrstuhl f. Biologische Chemie  |  Technische Universitaet Muenchen
Emil-Erlenmeyer-Forum 5  |  85350 Freising-Weihenstephan  |  Germany
Phone: +49 (0)8161 71-4351  |  Fax: -4352
http://www.wzw.tum.de/bc  |  eMail: ske...@tum.de

Re: [ccp4bb] Why MAD didn't work but SAD works well

[Bosch, Juergen]

Dear George,

if #4 is correct, shouldn't he be able to get good SIRAS using the peak dataset 
as HA and the last collected dataset as "native"

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Aug 20, 2013, at 5:42 PM, George Sheldrick wrote:

Dear Yafang,

If radiation damage is not a major problem, MAD should give you more
phase information than SAD, i.e. better maps, especially
at low resolution. If SAD works but MAD doesn't, there are several
possible explanations:

1. (most likely) your datasets are inconsistently indexed. This can
hapen in various ways depending on the Laue group and the unit-cell. If
you are using hkl2map the plots of the anomalous CC between the
different datasets are a good quick check. Some programs (e.g. the
current shelxc) will try to detect this and correct it automatically.

2. You have mixed up the wavelengths or labels of the datasets and so
the dispersive difference comes out with the wrong sign.

3. You have significant radiation damage and the RIP and dispersive
differences have opposite signs. Always measure the inflection
dataset last so that they reinforce each other rather than canceling.

4. You have severe radiation damage and only the first (peak) dataset is
usable.

Best wishes, George


On 08/20/2013 11:05 PM, Yafang Chen wrote:
Hi All,

I have three datasets of SeMet-incorporated protein at peak, infl and
high wavelength respectively. SAD with peak dataset works well to
solve the phase problem. However, MAD with all three datasets didn't
work at all. The completeness of all three datasets are more than 99%.
So I think radiation damage should not be a problem. Does anyone have
any idea about the possible reasons that MAD didn't work in this case?
Thank you so much for any of your help!

Best,
Yafang

--
Yafang Chen
Graduate Research Assistant
Mesecar Lab
Department of Biological Sciences
Purdue University
Hockmeyer Hall of Structural Biology
240 S. Martin Jischke Drive
West Lafayette, IN 47907


--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582

Re: [ccp4bb] non-diffracting xtals

[Bosch, Juergen]

Well said Petri,

also how much PEG3350 do you have in your conditions ? More than 25% ? I'm 
going after cryo-conditions at this point, you might want to replace your 
PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350.
Almost sounds as if no optimization of the original conditions was performed 
yet.

Plenty to do for you, also since you have some crystal use them for seeding 
into your new screens.

Jürgen

On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote:

Hi Petri

They are non-diffracting at the home source and they are cryo cooled. Like 
david suggested I guess ill try introducing a buffer as my condition does not 
have a buffer. it is ammonium acetate and PEG 3350.

Thanks for the encouragement !

Mahesh


On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula 
mailto:petri.kurs...@gmail.com>> wrote:
Hi,

non-diffracting on the home source or state-of-the-art synchrotron? Cryocooled 
or room-temperature? What happens if you change the buffer but keep your pH? 
etc etc...

For an important project, one should never ever give up.

Petri


---
Petri Kursula, PhD
project leader, adjunct professor
Department of Biochemistry & Biocenter Oulu, University of Oulu, Finland
Department of Chemistry, University of Hamburg/DESY, Germany
www.biochem.oulu.fi/kursula
www.desy.de/~petri/research
petri.kurs...@oulu.fi
---





On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju 
mailto:mxl1...@psu.edu>> wrote:

Hello people

I recently obtained hexagonal rod like crystals (150x50x20 um) which turned out 
to be non diffracting. What is the usual convention for cases like this ? do 
people usually give up on the condition or still try to optimize it ?

The crystals are also not very reproducible. I believe it is because of 
ammonium acetate in the condition causing fluctuations in the pH because of its 
volatility. Is there any way to work around such a problem ?

Thanks

Mahesh





..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Data processing - twinned xtals

[Bosch, Juergen]

tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine with 
most SSRL beamlines except of the 12-2 microfocus - but that might have been 
fixed in the meantime.

Jürgen

On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote:

for #2)

I'd suggest get some of those Mitigen loops that are titled. I assume you have 
hexagonal plates as crystals and you really want to shoot along the thin area 
of the crystal down the sixfold. With normal loops it's an art to get that 
crystal to sit upright in the loop but not impossible if you take smaller loops.

My longest axis collected was 420 Å to ~2 Å resolution by this method.

Jürgen

On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote:

This is clearly a case of a crystal with a very long unit cell; a case
which should be approached mindfully.

HKL2000 has a default search for indexing solutions such that diffraction
along the longest unit cell will be resolved, with the assumed spot size.

The problem with such diffraction has 2 aspects:
1) how to process the already collected data where the spots are close to
each other;
2) how to collect future data.

Ad 1) The best solution is to reduce the spot size, so the spots are
resolved. This may require an adjustment of spot size by a single pixel;
one should not only change spot radius, but also change the box size
between even and odd number of pixels in the box dimensions.

Just changing the spot radius changes the spot diameter by an even number
of pixels, so if one wants to change the spot diameter by one pixel, one
has to change the box size. This is the consequence of the spot being in
the center of the box.

Just during indexing, there is also a workaround by specifying the command
before indexing: longest vector followed by a number that defines the
upper limit of the cell size. This may help finding indexing, but will
create overlaps between spots during refinement and integration.

This dataset presents a problem of collecting data by rotating on the axis
perpendicular to the long unit cell. In consequence, the Image 1 has
essentially (barely differing in centroid position) overlapping spots, so
it would be hard to process them meaningfully by any program.

Ad. 2) What would be a better way to collect data in the future?


Hi CCP4 folks

I have a data set which is looks twinned ( see the image-1  - I zoomed on
to the image so that one can spot the twinning. Furthermore, the spots are
very smeary from ~ 30 - 120 degrees of data collection, see image 2) I
tried using HKL2000 and mosflm to process this data but i cannot process
it. I was wondering if anyone has any ideas as to how to process this data
or comments on whether this data is even useful. Also, I would really
appreciate if someone could share their experiences on solving twinning
issues during crystal growth

Thanks in advance !

Mahesh[image: Inline image 2][image: Inline image 3]



Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu<http://lupo.jhsph.edu/>





..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Data processing - twinned xtals

[Bosch, Juergen]

for #2)

I'd suggest get some of those Mitigen loops that are titled. I assume you have 
hexagonal plates as crystals and you really want to shoot along the thin area 
of the crystal down the sixfold. With normal loops it's an art to get that 
crystal to sit upright in the loop but not impossible if you take smaller loops.

My longest axis collected was 420 Å to ~2 Å resolution by this method.

Jürgen

On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote:

This is clearly a case of a crystal with a very long unit cell; a case
which should be approached mindfully.

HKL2000 has a default search for indexing solutions such that diffraction
along the longest unit cell will be resolved, with the assumed spot size.

The problem with such diffraction has 2 aspects:
1) how to process the already collected data where the spots are close to
each other;
2) how to collect future data.

Ad 1) The best solution is to reduce the spot size, so the spots are
resolved. This may require an adjustment of spot size by a single pixel;
one should not only change spot radius, but also change the box size
between even and odd number of pixels in the box dimensions.

Just changing the spot radius changes the spot diameter by an even number
of pixels, so if one wants to change the spot diameter by one pixel, one
has to change the box size. This is the consequence of the spot being in
the center of the box.

Just during indexing, there is also a workaround by specifying the command
before indexing: longest vector followed by a number that defines the
upper limit of the cell size. This may help finding indexing, but will
create overlaps between spots during refinement and integration.

This dataset presents a problem of collecting data by rotating on the axis
perpendicular to the long unit cell. In consequence, the Image 1 has
essentially (barely differing in centroid position) overlapping spots, so
it would be hard to process them meaningfully by any program.

Ad. 2) What would be a better way to collect data in the future?


Hi CCP4 folks

I have a data set which is looks twinned ( see the image-1  - I zoomed on
to the image so that one can spot the twinning. Furthermore, the spots are
very smeary from ~ 30 - 120 degrees of data collection, see image 2) I
tried using HKL2000 and mosflm to process this data but i cannot process
it. I was wondering if anyone has any ideas as to how to process this data
or comments on whether this data is even useful. Also, I would really
appreciate if someone could share their experiences on solving twinning
issues during crystal growth

Thanks in advance !

Mahesh[image: Inline image 2][image: Inline image 3]



Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Data processing - twinned xtals

[Bosch, Juergen]

Hi Mahesh,

TESTGEN START 1 END 120 ANGLE 0.5
moving the detector further back or using 2theta and collecting more frames.

When you started shooting at your image you should have checked for overlaps 
ahead of time.
I assume this to about 1.8 Å - maybe it would have been wiser to collect a 2.5 
Å dataset. Your corners have zero spots when you crank up the contrast, so you 
are not using all the available detector area, sure the completeness goes down 
but you might have avoided overlaps like this.

BUT not all is lost, if you dare going to XDS you might be able to squeeze 
sufficient information out of this data set.

Good luck,

Jürgen

On Aug 16, 2013, at 10:38 AM, Mahesh Lingaraju wrote:

Hi CCP4 folks

I have a data set which is looks twinned ( see the image-1  - I zoomed on to 
the image so that one can spot the twinning. Furthermore, the spots are very 
smeary from ~ 30 - 120 degrees of data collection, see image 2) I tried using 
HKL2000 and mosflm to process this data but i cannot process it. I was 
wondering if anyone has any ideas as to how to process this data or comments on 
whether this data is even useful. Also, I would really appreciate if someone 
could share their experiences on solving twinning issues during crystal growth

Thanks in advance !

Mahesh

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

[ccp4bb] Buccaneer question @Kevin

[Bosch, Juergen]

Hi Kevin et al.

Buccaneer IS a great program and I like it very much.

I have a testcase here for my X-ray class where a trimer is in the asymmetric 
unit, however all copies are different (not in sequence, but in space - think 
of Calmodulin for example).

There is no option to force Buccaneer to not use NCS right ? In this particular 
case the autobuilding is not performing as well as it could if it were not to 
use NCS.

I assume from the command line -ncsbuild is turning it on, however does not 
including this keyword turn off NCS building, or is your program so smart that 
it overrides the user wishes because it knows better ?

Thanks,

Jürgen

P.S. I can provide you with the SeMet phased dataset and the sequence for you 
to play with.
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Error running Pointless in CCP4-6.3 OSX (10.8)

[Bosch, Juergen]

Hi Sheena,

simple quickfix to your problem:
a) Run CAD and limit the output resolution on that mtz file so you don't run 
into the memory allocation error of pointless.
b) try phenix.xtriage also if it is not a CCP4 program

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu




On Aug 11, 2013, at 9:54 PM, Sheena McGowan (Med) wrote:

Dear all

We have been trying to run pointless on a large mtz file which seems to be 
allocating more than 1.5Gb of memory resulting in the following error:

The program run with command: /Applications/ccp4-6.3.0/bin/pointless
has failed with error message
pointless(507) malloc: *** mmap(size=1577435136) failed (error code=12)
*** error: can't allocate region
*** set a breakpoint in malloc_error_break to debug

We are using OSX 10.8.4 and believe that we need a 64bit version of pointless.

Looking at the pre release ftp site, we found the latest versions of pointless 
compiled for OSX 10.8, however they do not run.

dyld: Symbol not found: __ZNSt11range_errorD1Ev
  Referenced from: /Applications/ccp4-6.3.0/bin/pointless-1.8.7.osx10.8
  Expected in: /usr/lib/libstdc++.6.dylib
 in /Applications/ccp4-6.3.0/bin/pointless-1.8.7.osx10.8

Any help would be greatly appreciated (and any know if there is a upcoming 
release of a pre-compiled CCP4 OSX-64bit??)

Regards
Sheena



SHEENA McGOWAN | ARC Future Fellow
Department of Biochemistry and Molecular Biology
MONASH UNIVERSITY
Rm 243, Building 77, Wellington Rd, Clayton
Victoria, AUSTRALIA 3800
T + 61 3 9902 9309 | F + 61 3 9902 9500
E sheena.mcgo...@monash.edu | Skype s.mcgowan
W http://www.med.monash.edu.au/biochem/staff/mcgowan.html

Australian Society for Biochemistry and Molecular Biology (State 
Representative) http://www.asbmb.org.au/

39th Lorne Conference for Protein Structure and Function 
http://www.lorneproteins.org
February 9-13, 2014. Mantra Lorne, Lorne, AUSTRALIA

Re: [ccp4bb] Substrate/Ligand Induced Oligomerization of enzymes

[Bosch, Juergen]

FKBP ?
http://www.pnas.org/content/97/13/7096.full.pdf
Jürgen

On Aug 8, 2013, at 8:03 PM, Shiva Bhowmik wrote:

> Dear All,
> 
> I am looking for references and/or example of substrate or ligand induced 
> oligomerization of enzymes related to activation. 
> 
> Any help in this regard would be greatly appreciated.
> 
> Thanks.
> 
> Shiva

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] calculation of shape complementarity of different protein-ligand complexes

[Bosch, Juergen]

VROCS, www.eyesopen.com

Jürgen

On Aug 7, 2013, at 9:03 AM, Tobias Beck wrote:

Dear CCP4bb,

I would like to calculate the shape complementarity of several protein-ligand 
complexes (crystal structures with ligand available). This involves a set of 
different proteins and also different ligands. The ligands are similar in size, 
but not in chemical composition.

I have looked into the program sc (originally developed to calculate shape 
complementarity for protein-protein interfaces), but since the interfaces are 
rather small - as pointed out by Mike Lawrence - it might not be suitable for 
this type of problem.

Has anyone done something similar before? There are some mutants available, so 
it would be good to quantify the change in shape complementarity for different 
mutations/ligands for one protein, but also to be able to compare the different 
protein-ligand complexes to one another.

Thanks in advance,

Tobias.

--
___

Dr. Tobias Beck
ETH Zurich
Laboratory of Organic Chemistry
Wolfgang-Pauli-Str. 10, HCI F 322
8093 Zurich, Switzerland
phone:   +41 44 632 68 65
fax:+41 44 632 14 86
web:  http://www.protein.ethz.ch/people/tobias
___

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex

[Bosch, Juergen]

Hi Appu,

nothing really to worry about if you process your data with XDS or d*trek using 
3D profile fitting.
You still should be able to get something useful out of this data.
If you look at 90.png (I'm attaching a zoomed area of your image), you see your 
crystal is either split or you had two crystals in the loop. Look at the second 
row of reflections when starting from the beam stop position. Since the first 
and third row does not follow the same trend, I would think you have a second 
crystal in a slightly different orientation grown with the main crystal.
[cid:4DB7FA0D-B1B8-44C0-A7DD-B087B0E83914@sph.ad.jhsph.edu]
You should not try to index on all reflections, try getting one single lattice 
and ignore the other one. You will run into overlapping issues from the two 
lattices but you might still get good enough data to try MR when you process 
with XDS. With XDS you have the advantage of collecting all strong reflections 
over the whole dataset and then index on those.

You can download it from here:
http://xds.mpimf-heidelberg.mpg.de

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu


On Aug 1, 2013, at 9:44 AM, Appu kumar wrote:

Dear all ccp4 user,
 We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA 
complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A 
region but the whole data is completely spreaded to 2.8A. There are many spots 
which are very near to each other as if they were merged but closely placed. 
When we are trying to index the data, its not picking all the spots correctly 
and giving a unit cell dimension variable from 150 to 500A in one of the axis. 
Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried 
processing with HKL2000 but not able to index it., I am attaching four 4 images 
for every 90 degree frames collected. Please look at these images and give your 
valuable input regarding indexing problem.

your suggestions and support will be highly appreciated. Please guide me and 
help me sorting out the problem.

Thank you




<90.png><180.png><250.png><371.png><0.png>




<>

Re: [ccp4bb] Dose anyone see this ligand before?

[Bosch, Juergen]

Try here:
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=8031&loc=ec_rcs
download the sdf and use the sdf to pdb converter
Or I think phenix can read in sdf and convert them

Jürgen

On Jul 17, 2013, at 9:35 AM, Wei Feng wrote:

Dear all,
Thank you for your advices.
I had tried to use MPD and pyrophosphate etc to fix the density map but all of 
them were too small.
We guess that the molecular formula should be C8H18O2. So we search this 
formula in google and find two candidate molecules
1: http://flyingexport.en.ecplaza.net/dhad-99-5--137042-689140.html
2: http://en.m.wikipedia.org/wiki/Di-tert-butyl_peroxide
Could you tell me how to get the coordinate of these molecules?
Thank you for your time!
Wei





..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Dose anyone see this ligand before?

[Bosch, Juergen]

Yes that was what I was thinking as well, that's why the size-comparisson.

Jürgen

On Jul 16, 2013, at 11:56 AM, Oganesyan, Vaheh wrote:

It actually looks much like pyrophosphate! If your protein is phosphatase and 
the extra density is in vicinity of the active site it might be the remaining 
product of reaction.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, 
Juergen
Sent: Tuesday, July 16, 2013 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Dose anyone see this ligand before?

Special position ?
sulfate-size ?
Jürgen

On Jul 16, 2013, at 11:35 AM, Wei Feng wrote:


Dear all,
I found some redundant density in my structure beween two molecule. (see 
picture 1 and 2)
But I am not sure which ligand will be.
Dose everyone see this ligand before? If so, can you tell the PDB code or send 
me the struture file?
Thank you for your time!
Wei





..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu



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sender advising of the error in transmission and delete the original message 
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..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Dose anyone see this ligand before?

[Bosch, Juergen]

Special position ?
sulfate-size ?
Jürgen

On Jul 16, 2013, at 11:35 AM, Wei Feng wrote:

Dear all,
I found some redundant density in my structure beween two molecule. (see 
picture 1 and 2)
But I am not sure which ligand will be.
Dose everyone see this ligand before? If so, can you tell the PDB code or send 
me the struture file?
Thank you for your time!
Wei





..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Concentrating purified membrane protein

[Bosch, Juergen]

can you elute off the column with low pH ? Or EDTA if you don't want imidazole 
around ?

Jürgen

On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote:

Thanks everyone for your responses. I definitely plan to save the flowthrough 
so we'll see what happens. My protein has a His tag and I did consider doing an 
affinity step for concentration except I do not want to have imidazole for some 
functional assays that I need to carry out with the protein. Just occurred to 
me that I could simply dialyze out the imidazole after the affinity step.

Thanks again!
Raji


On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam 
mailto:r...@brandeis.edu>> wrote:
Hi Folks,

Sorry for the non-ccp4 post.

I have purified an 18kDa membrane protein and want to concentrate the protein 
from gel filtration fractions, which are in buffer containing 0.05% DDM (well 
above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane 
protein using a 100kDa MWCO concentrator but I am not sure if I can do the same 
without losing protein in the flowthrough. On the other hand, if use too low a 
MWCO for the concentrator, then I'm concerned that I may end up concentrating 
the DDM and end up with too much detergent in the final sample.

Any tips about how to concentrate my low MW protein without concentrating the 
DDM?

Many thanks.
Raji

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University




--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] XDS not picking spots correctly, please help!

[Bosch, Juergen]

Hi Fei,

I assume you have edited the XDS.INP file to include the initially found space 
group and the first refined cell ?
If not, then that would be my first choice to do. If that then still fails with 
the default parameters, then I would start fixing some values instead of 
refining all of them at the same time.
The keywords to look for are the following:
SPACE_GROUP_NUMBER=
UNIT_CELL_CONSTANTS=
REFINE(IDXREF)=
REFINE(INTEGRATE)=
REFINE(CORRECT)=

Once XDS successfully integrates your dataset coy GXPARM.XDS to XPARM.XDS and 
copy those values into the UNIT_CELL_CONSTANTS and rerun XDS.INP

And more details on each of those can be found here:
http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds/html_doc/xds_parameters.html

You should be able to take care of your problem as it doesn't sound severe.

Jürgen

On Jul 4, 2013, at 12:26 PM, Fei Li wrote:

> Dear all,
> 
> I recently collected some 10 degree wedges of data on a very small  
> membrane protein. The data processing so far is not working well. XDS  
> is the only software that process my data and give a space group and  
> cell parameters. However, I ended up with a merged dataset with low  
> completeness at low resolution. My data is not overloaded. This is a  
> very small protein shot with 5um mini-beam. I checked the FRAME.cbf  
> file and looks like XDS is not picking the spots at low resolution  
> correctly. It missed a good portion of my low res spots in every  
> datasets I checked and picked more at high res, which are weaker. Then  
> the indexing step will reject most of my spots for "too far away from  
> ideal position". imosflm was able to pick the spots correctly, but  
> does not index. I can send the FRAME.cbf file and the imosflm picture  
> if anyone thinks that will he useful to see. Any suggestion on how to  
> make XDS, or imosflm, correctly working for me is greatly appreciated.  
> Thanks a lot!
> 
> Best regards,
> 
> Fei
> 
> 
> Fei LI
> Graduate Assistant
> 310 Biochemistry Building
> Department of Biochemistry and Molecular Biology
> Michigan State University
> East Lansing, MI
> 48824

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

[ccp4bb] quick off topic question

[Bosch, Juergen]

Anybody out there who knows how many people are subscribed to the BB ?
Just curious. And no please don't reply all :-)

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Hi

[Bosch, Juergen]

The other obvious conclusion would be that dataset #3 is a different protein 
perhaps ?
How about pointless for your third dataset ?
Jürgen

On Jun 10, 2013, at 2:57 PM, Wei Shi wrote:

Hi all,
I was trying to solve the structure of a protein in several different datasets 
using xds and phenix. I could solve the structure from one dataset in space 
group P4. For another dataset, I could solve the structure using the monomer of 
the structure I got from the first dataset as search model and solve the 
structure in space group mC. For the third dataset, in IDXREF.LP, the space 
group of the highest symmetry is hp: 101.2, 101.3, 58.8, 90, 90, 120. According 
to Mathiews coefficient, 1 monomer is expected in the asymmetric unit. But I 
couldn't get the molecular replacement solution using the same method as for 
the second dataset. I also tried several other search models (eg. deletion of 
the potential flexible region in the search model) and tried to find the 
solution in all possible pointgroup. I also tried to process the data in oC 
(C222), the space group of the second highest symmetry in IDXREF.LP, but, still 
I could not get right molecular replacement solution.  I don't whether this 
means that there is a big conformational change for the structure in the third 
dataset or the space group I use is not right. Let me know if any of you would 
have any comments or suggestions for me. Thank you so much!

Best,
Wei



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Hi

[Bosch, Juergen]

Hi Eleanor,

C2 - this was XDS lingo or Bravais talk :-)

Jürgen


** LATTICE SYMMETRY IMPLICATED BY SPACE GROUP SYMMETRY **

BRAVAIS-POSSIBLE SPACE-GROUPS FOR PROTEIN CRYSTALS
 TYPE [SPACE GROUP NUMBER,SYMBOL]
 aP  [1,P1]
 mP  [3,P2] [4,P2(1)]
mC,mI[5,C2]
 oP  [16,P222] [17,P222(1)] [18,P2(1)2(1)2] [19,P2(1)2(1)2(1)]
 oC  [21,C222] [20,C222(1)]
 oF  [22,F222]
 oI  [23,I222] [24,I2(1)2(1)2(1)]
 tP  [75,P4] [76,P4(1)] [77,P4(2)] [78,P4(3)] [89,P422] [90,P42(1)2]
 [91,P4(1)22] [92,P4(1)2(1)2] [93,P4(2)22] [94,P4(2)2(1)2]
 [95,P4(3)22] [96,P4(3)2(1)2]
 tI  [79,I4] [80,I4(1)] [97,I422] [98,I4(1)22]
 hP  [143,P3] [144,P3(1)] [145,P3(2)] [149,P312] [150,P321] [151,P3(1)12]
 [152,P3(1)21] [153,P3(2)12] [154,P3(2)21] [168,P6] [169,P6(1)]
 [170,P6(5)] [171,P6(2)] [172,P6(4)] [173,P6(3)] [177,P622]
 [178,P6(1)22] [179,P6(5)22] [180,P6(2)22] [181,P6(4)22] [182,P6(3)22]
 hR  [146,R3] [155,R32]
 cP  [195,P23] [198,P2(1)3] [207,P432] [208,P4(2)32] [212,P4(3)32]
 [213,P4(1)32]
 cF  [196,F23] [209,F432] [210,F4(1)32]
 cI  [197,I23] [199,I2(1)3] [211,I432] [214,I4(1)32]

On Jun 10, 2013, at 4:51 PM, Eleanor Dodson wrote:

I don't really understand what your space group is? space group mC???
Eleanor
On 10 Jun 2013, at 19:57, Wei Shi wrote:

Hi all,
I was trying to solve the structure of a protein in several different datasets 
using xds and phenix. I could solve the structure from one dataset in space 
group P4. For another dataset, I could solve the structure using the monomer of 
the structure I got from the first dataset as search model and solve the 
structure in space group mC. For the third dataset, in IDXREF.LP, the space 
group of the highest symmetry is hp: 101.2, 101.3, 58.8, 90, 90, 120. According 
to Mathiews coefficient, 1 monomer is expected in the asymmetric unit. But I 
couldn't get the molecular replacement solution using the same method as for 
the second dataset. I also tried several other search models (eg. deletion of 
the potential flexible region in the search model) and tried to find the 
solution in all possible pointgroup. I also tried to process the data in oC 
(C222), the space group of the second highest symmetry in IDXREF.LP, but, still 
I could not get right molecular replacement solution.  I don't whether this 
means that there is a big conformational change for the structure in the third 
dataset or the space group I use is not right. Let me know if any of you would 
have any comments or suggestions for me. Thank you so much!

Best,
Wei



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Active site volume calculator

[Bosch, Juergen]

Hi Yuri,

http://fpocket.sourceforge.net
http://sts-fw.bioengr.uic.edu/castp/calculation.php

Just to name 2
Jürgen

On Jun 5, 2013, at 7:12 PM, Yuri Pompeu wrote:

> Dear BB,
> I am sorry for posting off-topic but it is hard not to ask when you know you 
> can get a good answer ;-)
> 
> I need to calculate the volume of several active sites. Nothing fancy, just a 
> number for comparison sake.
> I understand there are probably 10 different ways/programs and I would 
> appreciate some feedback.
> 
> cheers

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Helix forming proteins crystallizing as rings

[Bosch, Juergen]

Hi Joern,

have a look at these two papers (and probably KK is reading this too and can 
chime in with the right set of coordinates), 

Korotkov et al. Secretins: dynamic channels for protein transport across 
membranes. Trends Biochem Sci (2011) vol. 36 (8) pp. 433-43

Reichow et al. Structure of the cholera toxin secretion channel in its closed 
state. Nat Struct Mol Biol (2010) vol. 17 (10) pp. 1226-32

I recall in one of the structures an "open continuos helix" with 12 molecules 
forming a spiral in the crystal lattice. The EM reconstruction showed 12fold 
symmetry and when you "flatten" the ring from the crystal structure it fitted 
well into the EM structure.

Don't recall details at this point, but I think this is what you are looking 
for.

Jürgen


On Jun 5, 2013, at 7:44 AM, Joern Krausze wrote:

> Dear Tim,
> 
> the rings I mean are similar to the one in 4FC4 but to my understanding
> FocA does not form helices under physiological conditions.
> 
> I am sorry if I was not specific enough. I will give you more details. I
> am working on two proteins which are dimers in solution.
> 
> The first of them crystallizes as flat rings consisting of four or five
> dimers (the rings showing D4 and D5 point group symmetry, respectively),
> depending on the crystal form. We got three different crystal forms, one
> of which contains the D4 ring, two of which contain the D5 ring.
> 
> The second protein is homologous to the first and crstallizes in space
> group P1. Through the translational symmetry, the dimers form
> pseudo-infinite fibers. The crystal contacts are realized by residues
> homologous to the residues that form the interfaces in the rings of the
> first protein. These residues are conserved and hence seem to be of
> importance.
> 
> We now think that these proteins form helices under particular
> physiological conditions. We have (weak) biochemical data which support
> this assumption and it also makes sense mechanistically. We interpreted
> the crystal structures accordingly. We think that these helices are of the
> same width as the D4 or D5 rings but with a helical symmetry along the
> four- or fivefold axis instead of the purely rotational symmetry. We deem
> the rings of the first protein and the fiber of the second protein special
> cases of the helix: the rings are helices with a pitch of zero and the
> fiber would be a helix with zero turns (or a flat curvature).
> 
> A reviewer now does not really follow our argumentation and asked us to
> provide him with examples of proteins showing a similar behavior, i.e.
> normally forming helices but forming rings (or fibers) in the crystal.
> 
> Thanks to everyone for their help.
> 
> Best regards,
> 
> Joern
> 
> **
> Address:
> 
> Joern Krausze
> Molecular Structural Biology
> Helmholtz Centre for Infection Research
> Inhoffenstrasse 7
> 38124 Braunschweig
> Germany
> 
> Email:  joern.krau...@helmholtz-hzi.de
> Phone:  +49 (0)531 6181 7023 (office)
> +49 (0)531 6181 7020 (lab)
> **
> 
> On Wed, 5 Jun 2013, Tim Gruene wrote:
> 
>> -BEGIN PGP SIGNED MESSAGE-
>> Hash: SHA1
>> 
>> Dear Joern,
>> 
>> I am not sure this is what you have in mind: just yesterday I heard a
>> talk from Oliver Einsle about FocA ion channels - look at PDB ID 4FC4,
>> for example forming a (flat) pentamer, and let us know if this is what
>> you mean.
>> 
>> Best,
>> Tim
>> 
>> On 06/04/2013 05:24 PM, Joern Krausze wrote:
>>> Dear all,
>>> 
>>> can anyone provide me with examples for proteins that form a
>>> helical oligomer under physiological conditions and flat rings
>>> (basically a helix with a pitch of 0) in the crystal? I only know
>>> examples of the other way around.
>>> 
>>> Thank you in advance.
>>> 
>>> Joern
>>> 
>>> ** Address:
>>> 
>>> Joern Krausze Molecular Structural Biology Helmholtz Centre for
>>> Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany
>>> 
>>> Email:  joern.krau...@helmholtz-hzi.de Phone:  +49 (0)531 6181 7023
>>> (office) +49 (0)531 6181 7020 (lab)
>>> **
>>> 
>>> 
>>> 
>>> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7
>>> | 38124 Braunschweig | www.helmholtz-hzi.de Das HZI ist seit 2007
>>> zertifiziertes Mitglied im "audit berufundfamilie"
>>> 
>>> Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe,
>>> Bundesministerium für Bildung und Forschung Stellvertreter: Rüdiger
>>> Eichel, Abteilungsleiter Niedersächsisches Ministerium für
>>> Wissenschaft und Kultur Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf
>>> Richter, MBA Gesellschaft mit beschränkter Haftung (GmbH) Sitz der
>>> Gesellschaft: Braunschweig Handelsregister: Amtsgericht
>>> Braunschweig, HRB 477
>>> 
>> 
>> - --
>> - --
>> Dr Tim Gruene
>> Institut fuer anorganische Chemie
>> Tammannstr. 4
>> D-37077 Goettingen
>> 
>> GPG Key ID =

Re: [ccp4bb] visualizing protein-protein interface

[Bosch, Juergen]

Hi Tobias,
this may or may not work.
calculate a map for both proteins, normalize them, put them on an identical 
cell and subtract one map from another.
The program that can do this is called mapman *gosh* a non-CCP4 thing.

Good luck,

Jürgen

On Jun 4, 2013, at 10:18 AM, Tobias Beck wrote:

Dear all,

I would like to visualize the interface of a protein-protein complex. The 
complex formation is modulated by a ligand that upon binding alters the overall 
structure of one protein, which then in turn forms the complex with the second 
protein (structure of the complex is known). Ligand binding does not seem to 
induce a discrete conformational switch, rather a subtle alteration of the 
contact interface. The isolated structure of the first protein without the 
ligand is known as well.

In addition to depicting the structural difference in conventional ways I was 
thinking to draw some kind of stretched out topology map of the surface that is 
interacting - first the surface from the isolated protein, then the surface as 
it is found in the complex and then maybe some kind of difference map...

Does anybody have some suggestions how to do this? I would prefer to do some 
kind of stretched out surface (basically a rectangle of the region of interest) 
and show "mountains and valleys" and their change, but not sure if this is 
feasible.

Thanks for any advice!

Best wishes,

Tobias.

--
___

Dr. Tobias Beck
ETH Zurich
Laboratory of Organic Chemistry
Wolfgang-Pauli-Str. 10, HCI F 322
8093 Zurich, Switzerland
phone:   +41 44 632 68 65
fax:+41 44 632 14 86
web:  http://www.protein.ethz.ch/people/tobias
___

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] anomalous scattering server down?

[Bosch, Juergen]

seems to be down sorry.
you should be able to use crossec from ccp4
Jürgen

On Jun 1, 2013, at 6:47 PM, Edward A. Berry wrote:

Is Ethan Merritt's anomalous scattering page at:
http://www.bmsc.washington.edu/scatter/
down or moved, or  the firewall I'm behind is blocking it?

I want to check feasibility of a native-iron MAD experiment,
and I'm not very good at math.

thanks,
eab

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] atomic coloring for the color blind

[Bosch, Juergen]

Thank you Mark for the link, this is really helpful.
Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu


On May 31, 2013, at 9:14 PM, Mark van der Woerd wrote:

Professor Rice,

When publishing in NAR (Nucleic Acids Research), it was recommended that we use 
"colors friendly to the color blind". You can read about it here: 
http://jfly.iam.u-tokyo.ac.jp/html/color_blind/

It is quite nice that they went to the trouble of showing us "how they see it". 
And there are plenty of suggestions how to do better. In fact, it turns out 
that you need not stay away from red and green, but define them somewhat 
different and it will work better.

Ever since I made all my illustrations like this once, I try to do it every 
time, for any journal, just in case. I consult this page often, it is quite 
helpful to me. See especially near the bottom of the page "colors unambiguous 
both to color-blinds and non-color-blinds".

Hope this helps you too.

Mark



-Original Message-
From: Phoebe A. Rice mailto:pr...@uchicago.edu>>
To: CCP4BB mailto:CCP4BB@JISCMAIL.AC.UK>>
Sent: Fri, May 31, 2013 2:35 pm
Subject: [ccp4bb] atomic coloring for the color blind

I feel badly that one of my undergrads had trouble telling an O from a C in a 
pymol homework set because he's color blind. (The assignment involved telling 
me why the a GTP analog (GDPCP) wasn't hydrolyzed).
Is there a handy by-atom coloring scheme I can recommend that works for the 
red-green color blind?
  thanks,
  "Professor Rice"

++
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] how to make the crystal thicker

[Bosch, Juergen]

Instead of making the crystal thicker, why don't you make the beamsize smaller 
? Aka microfocus
If this is your cryo-condition then I would work on it a bit more or change the 
drop ratio of protein:reservoir so that you can use a higher PEG3350 
concentration in your reservoir. I would aim for >25% PEG3350.

What happens if you change the salt to something different ?
Have you tried an additive screen ?

Jürgen


On May 28, 2013, at 9:18 PM, 姜艳 wrote:

Dear professors,
I get my crystal in 0.1M Tris, PH7.5, 200mM (NH4)2SO4, and 20% PEG3350, 
however, it is very thin. From one side, the diffraction is perfect, about 
2.2A, but from the other side, diffraction is too bad, the spots look like a 
thread! Process cannot be done by HKL2000.
As top guns of this field, could you  give me some suggestion to make the 
crystal thicker? I will be grateful for your kind help.
Best,
Jiang Yan

Institute of Biophysics, Chinese Academy of Sciences
Beijing, Chaoyang District



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Organic solvents for ligand solubilisation

[Bosch, Juergen]

Hi Klaus,

- small molecular weight PEG's e.g. 200 instead of DMSO, has the advantage of 
also helping to cryo protect
- Methanol (only for dispensing the compound into wells) then allow to 
evaporate and simply add your cryo-protected crystals, the hope is that 
sufficient of your ligand goes into solution
- different method but also efficient, use your protein as "solubilizer" . 
Figure out the lowest concentration of DMSO under which your ligand is soluble 
(maybe 0.5%) in solution at say 50 µM then take your diluted protein and mix it 
with the ligand, then concentrate to the required concentration needed for 
crystallization

Jürgen

On May 23, 2013, at 9:36 AM, Klaus Fütterer wrote:

Dear CCP4BB followers,

We are currently trying to obtain ligand-bound complexes for one of our 
proteins by soaking and/or co-crystallisation. We have had prior success for 
this protein, but using  a different class of ligands. The new ligand (in DMSO) 
remains in solution (more or less) when mixed with the 
reservoir/cryoprotectant, and the diffraction pattern survives the soaking 
nicely. Annoyingly though,  all we see are density peaks that match the size of 
DMSO and become more pronounced when increasing [DMSO] (to help solubilisation 
of the ligand). Soaking times varied between minutes to 16 hours. Kd was 
measured ( ~ 10 uM) in the solution state.

We have tried pyridine (which keeps the ligand in solution, but kills 
diffraction in an instant), and DMF (which doesn't keep the ligand in solution 
when mixing with cryoprotectant).

I am wondering whether the community has suggestions for alternative organic 
solvents that have been used to solubilise hydrophobic ligands, and are 
reasonably gentle to the protein crystal.

Thank you.

Klaus


===

Klaus Fütterer, Ph.D.
Reader in Structural Biology
  Undergraduate Admissions

School of Biosciences   P: +44-(0)-121-414 5895
University of Birmingham   F: +44-(0)-121-414 5925
Edgbaston E: 
k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK   W: http://tinyurl.com/futterer-lab
===






..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] cryocrystals

[Bosch, Juergen]

I dare say indefinitely. Your crystal life expectancy drops rapidly though with 
somebody not remembering to fill up the dewar. Transferring crystals to pucks 
etc. is another problem. I had some crystals which I could not remember what 
they were (label broke off the cane) and we simply reshoot them after >1 year 
and figured out it was the usual suspect from the cell dimensions.

Jürgen


On May 22, 2013, at 8:38 AM, Careina Edgooms wrote:

Hi
Does anybody know how long one could store a crystal in liquid nitrogen for 
before it will no longer diffract well? I'm talking in the order of weeks to 
months...
careina

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] how to solve crystal structure using published EM structure

[Bosch, Juergen]

You can use it as an MR model in Molrep for example. Or are you asking where to 
get the actual EM data from ? That would be here: 
http://www.ebi.ac.uk/pdbe/emdb/

Jürgen

On May 18, 2013, at 8:32 PM, LISA wrote:

Hi All,

The EM  structure of a complex was published at 8A. If we can collect crystal 
data of this complex. How to get the phase of this crystal with the EM 
structure? Thank you.

Sincerely,

Lisa


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] disordered helix

[Bosch, Juergen]

This looks to me like a good attempt for GraphEnt. Give it a shot and you might 
be positively surprised.

Jürgen

Sent from my iPad

On May 13, 2013, at 21:26, "Dale Tronrud"  wrote:

>Sometimes a floppy bit of a protein is even more floppy in a
> particular crystal form.  Your maps do not appear to support your
> model of a helix in this location.  I would not build it unless
> maps based on later refinement show something reasonable in the
> omit map.  (Of course if you leave out the helix, all your maps
> will be omit maps.)
> 
>It is quite common to submit models to the PDB that do not
> contain all of the amino acids expected based on the sequence.
> If you can't see where the chain goes you certainly can't be
> expected to build it.
> 
> Dale Tronrud
> 
> On 05/13/2013 04:23 AM, atul kumar wrote:
>> I have attached the map and omit map(after deleting helix) images.
>> 
>> 2Fo-Fc(1 sigma)
>> 
>> Fo-Fc(3sigma)
>> 
>> On 5/13/13, Eleanor Dodson  wrote:
>>> Hard to say without seeing the maps and experimenting. My first check would
>>> be to set the NTD occupancies to 0.0 - refine the CTD alone, then look at
>>> the maps in COOT.
>>> 
>>> Or maybe let an automatic modelling building program such as Buccaneer try
>>> to rebuild the NTD section, with starting phases from the CTD.
>>> 
>>> Eleanor
>>> 
>>> 
>>> 
>>> 
>>> On 13 May 2013 09:04, atul kumar  wrote:
>>> 
 Dear all,
 
 I have solved the structure of my protein by molecular replacement at
 2.9A, with Rfactor and Rfree 18 and 22 respectively. Overall everything
 seems fine, its a two domain protein NTD and CTD, the NTD have high
 average
 B factor compared to CTD. A helix of NTD seems to be disordered, I tried
 different geometric weights but the refined structure does not seem to
 follow proper geometry for this helix. The B-factor of this helix is very
 high compared to overall B factor for NTD and omit map shows only some
 partial density in this region( off course not conclusive). All the
 homologous structure have helix in this region although with high
 B-factor.
 Should I submit the current pdb or need more refinement?
 
 thanks and regards
 
 Atul Kumar
 
>>> 

Re: [ccp4bb] off topic question regarding SPR

[Bosch, Juergen]

Hi Faisal,

whatever makes your protein happy is good. What type of experiment were you 
thinking of ? Protein-protein interaction or protein-small molecule interaction 
? In the latter case add some Tween20 or Brij-35 into the mix. A decent 
overview can be found under this webpage for what to consider when running your 
experiment. http://www.sprpages.nl
Since you are on this board, my assumption is you have crystallography-grade 
protein, which is an excellent starting point for SPR experiments. One thing to 
consider though is to perhaps include a glycerol correction, similar like a 
DMSO calibration curve, but that very much depends on what type of experiment 
you want to pursue.

Feel free to send me details off-board,

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu


On May 5, 2013, at 5:07 PM, Faisal Tarique wrote:

Dear all

i have a basic question regarding SPR experiment and that is: What is the 
recommended glycerol concentration in the protein sample for doing SPR 
experiment and getting the best possible result.? i have kept 10% Glycerol in 
my protein preparation..is it O.K ?

Thanx in advance

--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] need suggestions

[Bosch, Juergen]

Hi Afshan,

You mention:

The protein prep is same which was using to get the Hits.

So how old is your prep ? Was it stored at 4C or in LN2? Do you know if your 
protein is stable under the conditions you stored it ?

How long after purification did it take you to get those crystals ?

How old is your screen ? MES goes bad as well over time.

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://lupo.jhsph.edu

On Apr 16, 2013, at 5:21, "Afshan Begum"  wrote:

> The protein prep is same which was using to get the Hits.

Re: [ccp4bb] 2Theta data set solving problem

[Bosch, Juergen]

I thought imosflm recognizes automatically the 2theta offset ?

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://lupo.jhsph.edu

On Apr 9, 2013, at 7:51, "navin narayanan" 
mailto:navi...@gmail.com>> wrote:

Hello everybody,

We have a diffraction data set from home source with 2theta at 20deg. But we 
are not able to solve the data set using CCP4 suite. The software is picking up 
only the lower resolution data but not the higher resolution data points. Also 
we are not able to merge data two sets, one with 2theta at 0deg and the other 
with 2theta at 20deg. Is there anyway to solve the data. Any help will be truly 
appreciated. Thank you in advance.



Navin V Narayanan

Re: [ccp4bb] delete subject

[Bosch, Juergen]

There's a second side to that.
Reviewers who can't get enough data and request even more when you submit a 
decent paper with 18 pages of supplement for example.

Jürgen

On Mar 29, 2013, at 7:32 AM, Toufic El Arnaout wrote:

http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0053374


On Fri, Mar 29, 2013 at 4:09 AM, Eric Bennett 
mailto:er...@pobox.com>> wrote:
Scott,

I'm not sure I understand your last paragraph.  Once researchers have had their 
data pass peer review (which I interpret as meaning a journal has accepted it), 
how often do you think it happens that it does not immediately get published?

Just depositing data in the PDB, or posting it on a public web site, is not 
"meet[ing] the veracity of peer review".  There is something to be said for 
giving credit to the first people who have subjected their data to peer review 
and had the data pass that step, otherwise people will be tempted to just post 
data of dubious quality to stake a public claim before the quality of the data 
has been independently checked.  In a case where this initial public 
non-peer-reviewed posting is of unacceptable data quality, that would dilute 
credit granted to another person who later obtained good data.

An unfortunate number of problematic structures still sneak through peer 
review.  Relaxing quality review standards that must be passed before a 
scientist gets to claim credit for a discovery is a step backwards IMO.

Cheers,
Eric





On Mar 28, 2013, at 5:06 PM, Scott Pegan wrote:

Hey everyone,

Both Mark and Fred make some good points.  I totally agree with Nat (beat me to 
the send button).  Although in an ideal world with all the advancements in 
crowd sourcing and electronic media, one might think that posting data on a 
bulletin board might be considered marking one's turf and protect the scientist 
place in that pathway towards discoveries.  Regrettably, the current reality 
doesn't' support this case.  As structural biologists, we are still in the mode 
of first to publish gets the bulk of the glory and potentially future funding 
on the topic.

For instance, when I was in graduate school, the lab I was in had KcsA crystals 
at the same time as a couple of competing groups.  Several groups including the 
one I belong to had initial diffraction data.  One group was able to solve 
KcsA, the first K channel trans-membrane protein structure, first.  That group 
was led by Roderick Mackinnon, now a Noble Laureate partly because of this 
work.  Now imagine if one of Mackinnon's student would have put up the web 
their initial diffraction data and another group would have used it to assist 
in their interpretation of their own data and either solved the structure 
before Mackinnon, or at least published it prior.  Even if they acknowledged 
Mackinnion for the assistance of his data (as they should), Mackinnion and the 
other scientists in his lab would likely not have received the broad acclaim 
that they received and justly deserved.  Also, ask Rosalind Franklin how data 
sharing worked out for her.

Times haven't changed that much since ~10 years ago.  Actually, as many have 
mentioned, things have potentially gotten worse.  Worse in the respect that the 
scientific impact of structure is increasingly largely tide to the 
biochemical/biological studies that accompany the structure.  In other words, 
the discoveries based on the insights the structure provides.  Understandably, 
this increasing emphasis on follow up experiments to get into high impact 
journals in many cases increases the time between solving the structure and 
publishing it.  During this gap, the group who solved the structure first is 
vulnerable to being scoped.  Once scoped unless the interpretation of the 
structure and the conclusion of the follow up experiments are largely and 
justifiably divergent from the initial publications, there is usually a 
significant difficulty getting the article published in a top tier journal. 
Many might argue that they deposited it first, but I haven't seen anyone win 
that argument either.  Because follow up articles will cite the publication 
describing the structure, not the PDB entry.

Naturally, many could and should argue that this isn't they way it should be. 
We could rapidly move science ahead in many cases if research groups were 
entirely transparent and made available their discovers as soon as they could 
meet the veracity of peer-review.   However, this is not the current reality or 
model we operate in.  So, until this changes, one might be cautious about 
tipping your competition off whether they be another structural biology group 
looking to publish their already solved structure, or biology group that could 
use insights gathered by your structure information for a publication that 
might limit your own ability to publish. Fortunately, for Tom his structure 
sounds like it is only important to a pretty specific scientific question that 
many folks might not be work

Re: [ccp4bb] refinement protein structure

[Bosch, Juergen]

Hi Tom,

some suggestions for you:

You should calculate a selfrotation function and see if you can learn something 
from it.
You should definitely run Matthews Coefficient and see if you get a brilliant 
idea there

And congratulations to your first crystal structure it looks really great, for 
a starter this is the right resolution to play with and get exposed to all the 
programs used in the crystallographic community.

You got my other email off-the board but for the record keepers here, in case 
you were offended by my early April fools joke I do apologize. And the second 
part in the private email still holds true.

Jürgen

P.S. may I use your data in an X-ray workshop ?

On Mar 27, 2013, at 5:26 PM, Alexander Aleshin wrote:

But the amount of time spent on turning a protein into a publishable structural 
data is pretty much same, if not larger. There are no low hanging fruits any 
more.

On Mar 27, 2013, at 11:50 AM, Frank von Delft wrote:

Is it too much to dream that Tom has set a trail-blazing precedent and 
demonstrated to us all how unnecessary it is be anal about our oh-so-precious 
data and structures that in the year 2013 are almost completely useless without 
a huge dollop of other experimental data...?



On 27/03/2013 18:32, Anastassis Perrakis wrote:
I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!

Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Twinning problem

[Bosch, Juergen]

get the twin law and either refine with phenix.refine twin_law="-h,-k,l" or 
whatever it suggests, or just add into your Refmac script the line TWIN and it 
will figure out the twin law for you.

You can also detwin data but then you might be throwing away a lot of data. 
We've now had two cases with twin fractions close to 49% and they can 
definitely not be refined in a higher symmetry space group. One was P21 the 
other I222.

Jürgen


On Mar 26, 2013, at 10:45 AM, Liang Zhang wrote:

Hi, All,

I got a set of P2(or P21) data for MR. However, the Phenix-Xtriage indicated 
that it could be a pseudo-merohedral twinning. Does anyone know how to deal 
with such kind of twinning problem? Thanks.

Best,

Liang

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Isothermal titration calorimetry

[Bosch, Juergen]

Hi George,
this is probably a very stupid suggestion and you likely have tried it, but 
I'll suggest the obvious nevertheless.
What happens to your .nitc file when you rename it to .itc can you read it in 
Origin then ?

Jürgen


On Mar 24, 2013, at 6:39 AM, George Kontopidis wrote:

Chris,  indeed nanoITC  instrument analysis software is very robust and user
friendly (probable more friendly than microcal, GE).

Although when you need  to subtract  Q (heat)  values (from 2 or 3 blank
experiments) from your experimental data you cannot. NanoITC software  can
subtract  Q values only  from 1 blank experiment.
Also if you want to present  your data in a form of  heat/mol in Y
(vertical) axes  again you cannot. It presents  data in Y axes only in form
of heat/injection.
If you have found a way to extract 2 or 3 blank experiments from
experimental data or present data in form of heat/mol, please let me know it
will be very useful.

The main problem in the output files from nanoITC come with an extension
.nitc, by default.  Unfortunately Origin (that can do all the above) can
read only,  filenames with an extension .itc

Cheers,

George

-Original Message-
From: Colbert, Christopher [mailto:christopher.colb...@ndsu.edu]
Sent: Saturday, March 23, 2013 5:56 PM
To: George Kontopidis; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Isothermal titration calorimetry


George, would you please explain your comments?  We've found the TA
Instruments analysis software very robust and user friendly.

We have the low volume nanoITC from TA instruments and get equivalent #'s in
our comparison tests to the Microcal instrument.

Cheers,

Chris


--
Christopher L. Colbert, Ph.D.
Assistant Professor
Department of Chemistry and Biochemistry North Dakota State University P.O.
Box 6050 Dept. 2710 Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324





On 3/23/13 8:47 AM, "George Kontopidis" 
mailto:gkontopi...@vet.uth.gr>> wrote:

Keep in mind that output files from  nanoITC, TA instrument cannot be
red by Origin.  At some point you will need to analyse your data
further.

George

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Anastassis Perrakis
Sent: Saturday, March 23, 2013 12:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Isothermal titration calorimetry

It might be worth to consider the question more in detail.

Do you want to study thermodynamics of the interaction, or a KD would do?
If
the former, you need ITC. If the latter, and you want to study things
at the level of KD only, maybe investing on a plate reader,
thermophoresis, or some biosensor technology (spr or interferometry
based systems) should be considered.

Then, what interactions will you study with the ITC? In general, I
would agree that the lower sample volume is worth the nano options, but
depending on the typical systems under study, sometimes the gain on
sample quantity is not worth the money - while many times its worth it.

John is if course right that for studying specific systems as the one
he describes the 200 is great.

A.

Sent from my iPhone

On 23 Mar 2013, at 11:00, John Fisher 
mailto:johncfishe...@gmail.com>> wrote:

I would recommend the Microcal ITC 200, hands down. Not only is it an
amazing instrument with the optional automated sample loader (which is
worth every penny), but we were able to do experiments (multiple) using
FULL-LENGTH p53 binding to a weak cognate protein. I believe this was
the first time ITC was ever used with full length p53, as it is so
labile and just loves immediately to oligomerize. Sample sizes pay for
the instrument.
Best,
John

John Fisher, M.D./PhD
St. Jude Children's Research Hospital Department of Oncology
Department of Structural Biology
W: 901-595-6193
C: 901-409-5699

On Mar 23, 2013, at 4:45 AM, Sameh Soror 
mailto:shamd...@googlemail.com>>
wrote:

Dear All,


I am sorry for the off topic question. I am going to buy ITC to
study
protein-protein & protein-ligand interactions

I am comparing microcal, GE and nanoITC, TA instrument..
any suggestions, recommendations, good experiences or bad experiences.
is
there a better system.


Thank in advance for the help.


Regards


Sameh

--
Sameh Soror

Postdoc. fellow




..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] ionic interaction inside a protein

[Bosch, Juergen]

Try PDBSum
Jürgen

On Mar 23, 2013, at 9:34 AM, Faisal Tarique wrote:

Dear all

I am working on a thermostable protein and i have read that the stability to 
high temperature is due to  various ionic interactions among the amino acid 
residues of the protein itself..I request you all to tell me any web server 
which can show all the ionic interactions between amino acid residues in my  
protein structure by just uploading its coordinate file.in a 
nut shell i want to identify those residues in my protein which are taking part 
in ionic interaction.

--
Regards

Faisal
School of Life Sciences
JNU

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Map Alignment

[Bosch, Juergen]

If I understand your question correctly, you have a couple of atoms which you 
could align to get the rotation & translation then you can use these values 
with maprot (CCP4) or mama (USF) to actually superimpose maps.

Jürgen

On Mar 21, 2013, at 11:29 PM, Chen Zhao wrote:

Dear all,

Does anybody know some softwares for aligning electron density maps?

I tried transforming map by SQL model fit extension in COOT, which turned out 
to be not working: the map it transformed is the one supposed to be fixed. If I 
switch the moving model with the reference model, I only got some error 
messages.

I also tried the "Superpose maps" utility in PHENIX, however, since they are 
nucleic acid structures, it seems that the sequences cannot be recognized.

Thank you very much!

Best,
Chen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] suitable buffer for CD studies

[Bosch, Juergen]

Yep,

mostly you should stay away from Tris as this is the worst buffer system when 
playing with temperature changes. Tris for example has a ∆pKa/10˚C -0.31

Good, N.E. (1986) Biochemistry 5, 467

Jürgen

P.S. @Matthew, was this what you meant by "the Good buffers often not" ? or 
just a coincidence ?

On Mar 20, 2013, at 9:57 PM, Matthew Merski wrote:

One of the other things you need to be concerned about with thermal melts is 
the change in buffer pKa as temperature varies (I seem to remember this being 
called the "beta" factor).  Phosphate is used for CD melts regularly because 
its pKa is fairly invariant with temperature.  (A good reference is "Data for 
Biochemical Research" by Dawson, Ch. 18).  Acetate also shares this invariance 
but the Good buffers often do not.  This is of course a concern with the Spyro 
Orange experiment as well.


Matthew Merski
Shoichet Group
UCSF


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] suitable buffer for CD studies

[Bosch, Juergen]

Why not thermal denaturation in the presence of Sypro Orange and a realtime PCR 
machine ?

Crowther et al. Use of thermal melt curves to assess the quality of enzyme 
preparations. Anal Biochem (2010) vol. 399 (2) pp. 268-275

Or (shameless advertisement):
Hain et al. Structural characterization and inhibition of the Plasmodium 
Atg8-Atg3 interaction. Journal of Structural Biology (2012) vol. 180 (3) pp. 
551-62

2.9.2. Thermal shift assay

Mutant and wild type proteins were tested at 1 mg/mL in a reaction containing 
30 lL of protein and 1 lL of a 1:30 dilution of SYPROÒ orange dye. Fluorescent 
measurements were done in triplicates in a CFX96 thermal cycler (BioRad) from 
20 to 80 °C over a period of 60 min and the melting temperature was determined 
from the maximum of the first derivative of the melting curve.

You will need less protein compared to the CD melt.

Jürgen

On Mar 20, 2013, at 8:56 PM, Kavestri wrote:

Hi Harsh

Something like sodium borate at pH 9.0 could be an alternative to phosphate 
buffers. If you are looking at thermal unfolding above 220nm, then the choice 
of buffer is less critical as many buffers and additives are problematic only 
below 200nm.

If your samples require high salt concentrations, I routinely use NaF as an 
alternative. It is transparent in the wavelength range relevant for far UV CD 
spectral collection.

Kavestri Y.
Kingston Laboratory - Structural Biology Group
University of Auckland

On 20/03/2013, at 7:59 PM, Harsh Bansia 
mailto:spideysp...@gmail.com>> wrote:


Sorry for a simple and non-CCP4 question.


I have determined the structures of three different mutants of a thermostable 
protein by X-ray crystallography method. I feel that Mg2+ has a role in protein 
stability.


So I want to perform a thermal denaturation study by CD spectroscopy both in 
presence and absence of Mg2+ ion.

In this regards, what should be the suitable buffer for CD studies? May I use 
PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it 
advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion 
that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized 
and have theoretical pI =4.56 and maximum activity at pH 8.4.


Thanking you in advances,

harsh

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] first use of synchrotron radiation in PX

[Bosch, Juergen]

Thank you James, you should write a History book about the modern x-ray times.
Or better make one of those movies you are famous for.

Jürgen

On Mar 16, 2013, at 10:46 AM, James Holton wrote:

The first report of shooting a protein crystal at a synchrotron (I
think) was in 1976:
http://www.pnas.org/content/73/1/128.full.pdf
that was rubredoxin

The first PDB file that contains a "SYNCHROTRON=Y" entry is 1tld
(trypsin), which was deposited in 1989:
http://dx.doi.org/10.1016/0022-2836(89)90110-1
But the structure of trypsin was arguably already "solved" at that time.

Anomalous diffraction was first demonstrated by Coster, Knoll and Prins
in 1930
http://dx.doi.org/10.1007/BF01339610
this was 20 years before Bijvoet.  But not with a synchrotron and
definitely not with a protein

The first protein to be solved using anomalous was crambin in 1981:
http://dx.doi.org/10.1038/290107a0
but this was not using a synchrotron

The first demonstration of MAD on a protein at a synchrotron was a Tb
soak of parvalbumin in 1985
http://dx.doi.org/10.1016/0014-5793(85)80207-6
but one could argue that several parvalbumins were already known at that
time.

The first MAD structure from native metals was cucumber blue copper
protein (2cbp) in 1989
http://dx.doi.org/10.1126%2Fscience.3406739

The first "new" structure using MAD, as well as the first SeMet was
ribonuclease H (1rnh) in 1990
http://dx.doi.org/10.1126/science.2169648

If anyone knows of earlier cases, I'd like to hear about it!

-James Holton
MAD Scientist

On 3/13/2013 7:38 AM, Alan Cheung wrote:
Hi all - i'm sure this many will know this : when and what was the
first protein structure solved on a synchrotron?

Thanks in advance
Alan



..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] [ccp4bb] validating ligand density

[Bosch, Juergen]

Going back to the initial question.
I would recommend looking at AFITT
http://www.eyesopen.com/afitt

Works like a dream (in certain cases).

Jürgen

P.S. I wish I had some stocks from them but I don't
..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Mar 12, 2013, at 11:25 AM, 
mailto:herman.schreu...@sanofi.com>> 
mailto:herman.schreu...@sanofi.com>> wrote:

You are the one who should judge your statement, but it looks plausible to me.

Now that I think of it: why do we need referees if every scientist should judge 
their own hypothesis? Publication will be a lot faster if we no longer need to 
heed the remarks of some grumpy referees and send in revision after revision. 
Also the number of publications will increase significantly if every scientist 
is allowed to judge their own papers!

HS


From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu]
Sent: Tuesday, March 12, 2013 4:14 PM
To: Schreuder, Herman R&D/DE
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] validating ligand density

Dear Jacob,
You are overinterpreting, the statement is about judging, not proving a 
hypothesis. I am sure Mr. Edwards judged his statement to be ok.

I guess there is a good likelihood that you are right, but who am I to judge?

JPK





Herman


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob 
Keller
Sent: Tuesday, March 12, 2013 3:44 PM

To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] validating ligand density

One final quote that is not in the twilight paper summarizes it nicely:

"The scientist must be the judge of his own hypotheses, not the
statistician."
 A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept
of likelihood and its application to scientific inference , p. 34.

There must be a lot of thinking behind this statement--while it seems 
plausible, it seems far from proven prima facie. Also, it assumes that the 
scientist is not a statistician.

Jacob







Btw, the book is good reading.

Best, BR

-Original Message-
From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie
Joosten
Sent: Tuesday, March 12, 2013 10:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] validating ligand density

Dear Srinivasan,

Although the Twilight program can only look at deposited PDB entries, the
tips about ligand validation in the paper are very useful. I suggest you
start from there.
You can use EDSTATS in CCP4 to get real-space validation scores. Also look
at the difference map metrics it gives (and the maps themselves of course),
they will tell you whether you misidentified your ligand. Occupancy
refinement in Refmac can also help you: if the occupancy drops a lot
something is wrong. That can be partial binding (not that much of a problem)
or worse, a ligand that isn't there. By the way,  I've been playing with
that recently and some ligands/hetero compounds in the PDB were so
incredibly 'not there' that Refmac would crash (that bug seems to be fixed
in the latest version).

HTH,
Robbie

> -Original Message-
> From: CCP4 bulletin board 
> [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> R.Srinivasan
> Sent: Monday, March 11, 2013 23:03
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] validating ligand density
>
> Hello all,
>
> We co-crystallized an inactive variant of our enzyme in
> the
presence of
> substrate and have determined the structure at 1.85A.
>
> Now, we want to validate the fitting of the ligand into
> the
electron
> density. We tried validating using the difference map (2Fo-Fc) after
refining
> the structure without the ligand. But, it is still a bit inconclusive
> if
the density
> fits the ligand.
>
> It would be very kind to know if there are tools for
validating this
> electron density. We were excited about twilight but turns out it can
> only
be
> used with deposited structure.
>
>
> We will appreciate your help and suggestions.
>
>
> Many thanks,
> Srinivasan



--
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***



--
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org<

Re: [ccp4bb] How to compare B-factors between structures?

[Bosch, Juergen]

Yep, I agree calculate the average B per structure and divide each B by this 
value, then multiply it by any value that is reasonable so you can visualize 
color differences :-)
Jürgen

On Mar 4, 2013, at 2:16 PM, Jacob Keller wrote:

You only entertain addition+subtraction--why not use multiplication/division to 
normalize the b-factors?

JPK

On Mon, Mar 4, 2013 at 2:04 PM, James Holton 
mailto:jmhol...@lbl.gov>> wrote:
Formally, the "best" way to compare B factors in two structures with different 
average B is to add a constant to all the B factors in the low-B structure 
until the average B factor is the same in both structures.  Then you can 
compare "apples to apples" as it were.  The "extra B" being added is equivalent 
to "blurring" the more well-ordered map to make it match the less-ordered one. 
Subtracting a B factor from the less-ordered structure is "sharpening", and the 
reason why you shouldn't do that here is because you'd be assuming that a 
sharpened map has just as much structural information as the better diffracting 
crystal, and that's obviously no true (not as many spots).   In reality, your 
comparison will always be limited by the worst-resolution data you have.

Another reason to add rather than subtract a B factor is because B factors are 
not really "linear" with anything sensible.  Yes, B=50 is "more disordered" 
than B=25, but is it "twice as disordered"? That depends on what you mean by 
"disorder", but no matter how you look at it, the answer is generally "no".

One way to define the "degree of disorder" is the volume swept out by the 
atom's nucleus as it "vibrates" (or otherwise varies from cell to cell).  This 
is NOT proportional to the B-factor, but rather the 3/2 power of the B factor.  
 Yes, 3/2 power.  The value of "B", is proportional to the SQUARE of the width 
of the probability distribution of the nucleus, so to get the volume of space 
swept out by it you have to take the square root to get something proportional 
the the width and then you take the 3rd power to get something proportional to 
the volume.

An then, of course, if you want to talk about the electron cloud (which is what 
x-rays "see") and not the nuclear position (which you can only see if you are a 
neutron person), then you have to "add" a B factor of about 8 to every atom to 
account for the intrinsic width of the electron cloud.  Formally, the B factor 
is "convoluted" with the intrinsic atomic form factor, but a "native" B factor 
of 8 is pretty close for most atoms.

For those of you who are interested in something more exact than "proportional" 
the equation for the nuclear probability distribution generated by a given B 
factor is:
kernel_B(r) = (4*pi/B)^1.5*exp(-4*pi^2/B*r^2)
where "r" is the distance from the "average position" (aka the x-y-z 
coordinates in the PDB file).  Note that the width of this distribution of 
atomic positions is not really an "error bar", it is a "range".  There's a 
difference between an atom actually being located in a variety of places vs not 
knowing the centroid of all these locations.  Remember, you're averaging over 
trillions of unit cells.  If you collect a different dataset from a similar 
crystal and re-refine the structure the final x-y-z coordinate assigned to the 
atom will not change all that much.

  The full-width at half-maximum (FWHM) of this kernel_B distribution is:
 fwhm = 0.1325*sqrt(B)
and the probability of finding the nucleus within this radius is actually only 
about 29%.  The radius that contains the nucleus half the time is about 1.3 
times wider, or:
r_half = 0.1731*sqrt(B)

That is, for B=25, the atomic nucleus is within 0.87 A of its average position 
50% of the time (a volume of 2.7 A^3).  Whereas for B=50, it is within 1.22 A 
50% of the time (7.7 A^3).  Note that although B=50 is twice as big as B=25, 
the half-occupancy radius 0.87 A is not half as big as 1.22 A, nor are the 
volumes 2.7 and 7.7 A^3 related by a factor of two.

Why is this important for comparing two structures?   Since the B factor is 
non-linear with disorder, it is important to have a common reference point when 
comparing them.  If the low-B structure has two atoms with B=10 and B=15 with 
average overall B=12, that might seem to be "significant" (almost a factor of 
two in the half-occupancy volume) but if the other structure has an average B 
factor of 80, then suddenly 78 vs 83 doesn't seem all that different (only a 
10% change).  Basically, a difference that would be "significant" in a 
high-resolution structure is "washed out" by the overall crystallographic B 
factor of the low-resolution structure in this case.

Whether or not a 10% difference is "significant" depends on how accurate you 
think your B factors are.  If you "kick" your coordinates (aka using "noise" in 
PDBSET) and re-refine, how much do the final B factors change?

-James Holton
MAD Scientist


On 2/25/2013 12:08 PM, Yarrow Madrona wrote:
Hello,

Does anyone know a good method to co

Re: [ccp4bb] B factor of the loop

[Bosch, Juergen]

yes - but keep in mind your protein is in the context of the crystal lattice, 
so flexible regions in solution are likely to be stabilized in the crystal 
lattice. So if you color by B also look at the symmetry mates.
And you should also submit both structures to the TLSMD server and look at 
those results.
http://skuld.bmsc.washington.edu/~tlsmd/

Jürgen

On Mar 3, 2013, at 4:35 PM, Faisal Tarique wrote:

> Dear all
> 
> Can B factor in the crystal structure be the criteria to look into the 
> flexibility of a region or domain.? Also if  two structures are at different 
> resolutions. 
> 
> Faisal
> -- 
> 

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] off topic question BIAcore problem

[Bosch, Juergen]

Well that looks pretty real then. You might have wrong concentrations in one or 
the other experiment perhaps hence the difference.

Jürgen

On Feb 20, 2013, at 5:45 PM, xianchi dong wrote:

Thanks a lot for the kind reply.

I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I 
have different truncations of my protein which behaved similarly.
I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is 
about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used 
was from 100 nM to 5 nM. I  used a control buffer which can disrupt the 
binding. In that buffer, no binding was observed.

I also used fluorescence anisotropy to measure the Ki of the interaction which 
showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay.


On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong 
mailto:dongxian...@gmail.com>> wrote:
Dear all,

Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?

Thanks in advance.

Xianchi Dong
Research Fellow
Children's Hospital Boston
Harvard Medical School


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] off topic question BIAcore problem

[Bosch, Juergen]

I assume you use CM5 chips ?
I further assume you run at pH 7.5 perhaps ? What's the pI of your analyte ? > 
7.5 ? Do you get significant binding to your reference cell under the 
conditions you are running ?
You might get rebinding to your negatively charged surface and the dissociation 
you are measuring might not really be correct (masked through rebinding)
Check that first I would say.
You can measure low picomolar dissociations. I recently had one with ~80 pM.

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Feb 20, 2013, at 12:03 PM, xianchi dong wrote:

Dear all,

Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?

Thanks in advance.

Xianchi Dong
Research Fellow
Children's Hospital Boston
Harvard Medical School

Re: [ccp4bb] Mac mini advice

[Bosch, Juergen]

I assume nobody of you is running an actual Osx server ? I mean the upgrade to 
a full server version of the commonly distributed normal Osx releases ?

I have not done it yet but I do think many of the issues mentioned regarding 
NFS/NIS could be addressed there. Regarding the missing macpro upgrades I 
expect to see new machines with thunderbolt connectivity in the next 4 months. 
And I will buy my third macpro then to run it as a true server.

Jürgen 

Sent from my iPad

On Jan 23, 2013, at 5:21, "Peter Keller"  wrote:

> On Wed, 2013-01-23 at 01:54 -0700, James Stroud wrote:
>> On Jan 22, 2013, at 11:20 PM, Nat Echols wrote:
>>> The real difficulty is integrating Macs into a
>>> Linux-centric environment, for example configuring NFS, NIS, etc.
>> 
>> That's because NFS and NIS are antiquities left over from the days of
>> mainframes. Distributed file systems and user information databases
>> are designed for an environment of many workers and few machines, when
>> the typical graphics workstation cost $50,000. These days, we argue
>> whether to spend an extra $200 on a $500 computer. We have moved to a
>> new paradigm: many workers with many more machines, with each machine
>> having essentially mainframe levels of storage and computing power.
> 
> Technically there is something in what you say as a pattern for
> day-to-day work (for some people, although not all), but I think that
> describing the debate in terms of modern vs. antiquated is missing the
> point completely. The real difference between local vs. centralised
> storage is to do with responsibility for the hardware and the data that
> it contains.
> 
> Local workstation storage is OK for the following kinds of cases:
> 
> (i) the data that are stored locally have no value, so it doesn't matter
> if they are lost (either through hardware failure, misbehaving software
> or accidental deletion).
> 
> (ii) the user has the expertise and the time to set up and maintain a
> strategy for recovering data that are lost from local disks
> 
> (iii) the institution that the user works for allows the user to include
> data on local workstation disks in the institution's regular backup
> operations
> 
> When none of these apply, there is a real, contemporary case for using
> something like NFS, where the storage is centrally maintained and backed
> up. The cost of storage has fallen of course, but what that means is
> that the real questions now are about the value of the data. In some
> fields, you could store your entire career's data on a few USB memory
> sticks, but I doubt that many people would want to do that without
> having made other copies somewhere else, and the same applies to local
> workstation storage too :-).
> 
> There are other considerations in favour of connecting a workstation to
> networked services: if you use more than one machine it can be an
> incredible pain to be constantly moving data around from one to the
> other, and to keep track of what the authoritative versions are. Having
> independent, local user id's and passwords on every workstation can also
> cause difficulties. I could go on
> 
>> In other words, instead of NFS, you should run git.
> 
> This is simply not an option for many crystallographers, who do not have
> a background in software development or data management. Advocating and
> supporting git (or indeed any content/version management system) for
> those kind of users is a losing battle: they see it as an unnecessary
> complication to their daily work, and will avoid using it as far as they
> can.
> 
> Regards,
> Peter.
> 
> -- 
> Peter Keller Tel.: +44 (0)1223 353033
> Global Phasing Ltd., Fax.: +44 (0)1223 366889
> Sheraton House,
> Castle Park,
> Cambridge CB3 0AX
> United Kingdom

Re: [ccp4bb] Mac mini advice

[Bosch, Juergen]

> Any reason for the Mac Mini over the iMac

A zalman monitor ir can you hook up a second monitor in stereo mode to your 
iMac ?

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://lupo.jhsph.edu

On Jan 22, 2013, at 15:13, "Antony Oliver"  wrote:

> Hi Cara, 
> 
> Any reason for the Mac Mini over the iMac? - presumably as you've got a 
> suitable monitor / keyboard already? 
> 
> We're pretty much exclusively iMac of late (ditched the towers) and finding 
> them absolutely fine for both fairly intensive jobs (refinement) and 
> visualisation/building (Coot / PyMOL). 
> 
> We working with the quad-core i7's - but a lower spec is probably ok too - 
> just boost the memory if you can. 
> 
> With regards, 
> 
> Tony. 
> 
> Sent from my iPhone
> 
> On 22 Jan 2013, at 18:00, "Cara Vaughan"  
> wrote:
> 
>> Dear CCP4BB
>> 
>> I'm thinking about buying a Mac Mini and was looking for advice from people 
>> who have used these for crystallography.
>> 
>> We don't need the computer to do serious number-crunching as we have 
>> back-end servers that can do this for us, so it is primarily for running 
>> coot for model building, etc. and low intensity crystallography jobs.
>> 
>> I've seen from the archive that some people do use the Mac Mini for 
>> crystallography and I've got two questions:
>> 1. Do I need the Quad core or is a Dual core processor enough?
>> 2. Is the intergrated Intel HD graphics card OK for crystallography 
>> requirements?
>> 
>> All the best,
>> Cara.
>> 
>> 
>> Cara Vaughan
>> Lecturer in Structural Biology
>> Institute of Structural and Molecular Biology
>> Birkbeck College and UCL
>> London UK
>> 
>> 
>> This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] Mac mini advice

[Bosch, Juergen]

Current Mac minis outperform my 2009 models with which I am still happy. So 
dual core I guess would be sufficient no need for upgrade on graphics card.

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://lupo.jhsph.edu

On Jan 22, 2013, at 11:59, "Cara Vaughan"  
wrote:

> Dear CCP4BB
> 
> I'm thinking about buying a Mac Mini and was looking for advice from  
> people who have used these for crystallography.
> 
> We don't need the computer to do serious number-crunching as we have  
> back-end servers that can do this for us, so it is primarily for  
> running coot for model building, etc. and low intensity  
> crystallography jobs.
> 
> I've seen from the archive that some people do use the Mac Mini for  
> crystallography and I've got two questions:
> 1. Do I need the Quad core or is a Dual core processor enough?
> 2. Is the intergrated Intel HD graphics card OK for crystallography  
> requirements?
> 
> All the best,
> Cara.
> 
> 
> Cara Vaughan
> Lecturer in Structural Biology
> Institute of Structural and Molecular Biology
> Birkbeck College and UCL
> London UK
> 
> 
> This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] a challenge

[Bosch, Juergen]

"What is the best procedure to use for weak anomalous signal"

That opens up the can of worms which I'm happy to jump into.
We've had very good success in the years 2003-2009 with shelx for finding sites 
(sometimes more than 1 trials) then force feeding them to sharp for phase 
improvement. We should also say most of the times in particular in the more 
difficult cases xds made the difference in detectable anomalous signal.

And no we still have not published this. With we I mean Marc Robien and myself 
during our SGPP times.

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://lupo.jhsph.edu

On Jan 14, 2013, at 3:13, "James Holton"  wrote:

> What is the best procedure to use for weak anomalous signal

Re: [ccp4bb] Coot's hidden talents!

[Bosch, Juergen]

Surprised Mercedes didn't sued him for that :-)

@Mr. Emsley I assume you soon will be Sir Emsley after that marvelous 
painting.mentioned in Harry's email.

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Jan 11, 2013, at 10:21 AM, Johan Turkenburg wrote:

Friday afternoon Good time to look at some artistic
representations of protein molecules. This is pretty amazing:

http://www.julianvossandreae.com/

Johan

On 11 January 2013 14:26, Derek Logan  wrote:
To be honest I preferred this more homespun work:

http://www.guardian.co.uk/science/gallery/2013/jan/10/research-as-art-competition-in-pictures?INTCMP=SRCH#/?picture=402066318&index=3

Somewhat reminiscent of Byron's Bender models:

http://www.umass.edu/microbio/rasmol/history.htm#bender

/Derek

On 11 Jan 2013, at 13:08, Mark J van Raaij  wrote:

if you zoom in 10exp9-10exp10 times (lots of cmd-+ in macosx), you can see the 
amino acids.

tried to check the accuracy; however, I couldn't find the mtz file to display 
the density...does this journal not enforce depositing the data?

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij



On 11 Jan 2013, at 11:47, Harry Powell wrote:

Hi

Just noticed this -

http://www.bbc.co.uk/news/uk-20978904

do we know the artist? He has just moved to Cambridge...

Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, 
Cambridge, CB2 0QH
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing)









--
Dr. Johan P. Turkenburg X-ray facilities manager
York Structural Biology Laboratory
University of York   Phone (+) 44 1904 328251
York YO10 5DD   UK  Fax   (+) 44 1904 328266

Note new email address johan.turkenb...@york.ac.uk

Re: [ccp4bb] Symmetry operator

[Bosch, Juergen]

coot: show symmetry molecules, then save symmetry molecule and you have your 
tetramer most likely

Jürgen

On Jan 10, 2013, at 7:48 PM, james09 pruza wrote:

Hi,

Which program outputs the symmetry operator (rotation and translation)? I have 
a dimer in the asymmetric unit and need to know the symmetry operator to get a 
tetramer, the active molecule.
James


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Who invented PDB format?

[Bosch, Juergen]

bash or any other shell script using http://www.ccp4.ac.uk/html/coordconv.html
Probably moleman2 http://xray.bmc.uu.se/usf/moleman_man.html can handle your 
request too.

Jürgen

On Jan 7, 2013, at 12:48 AM, Teri Arman wrote:

Fractional Coordiantes to Orthogonal Coordinates and Vice Versa

Hi, I need help, how can I make coordiates of 100 of PDBs of different space 
groups to fractional coordiantes and vice versa. I do not find CCP4 do it?  A 
program of fortran or C codes with possible suggestion may be helpful.
Thank you.
TA


On Sun, Jan 6, 2013 at 2:27 PM, George Sheldrick 
mailto:gshe...@shelx.uni-ac.gwdg.de>> wrote:
Chemical crystallographers have always used fractional coordinates, it makes it 
so
much easier to handle symmetry, special positions etc. But if the PDB hadn't
used orthogonal coordinates, bioinformatics might never have taken off.

George


On 01/06/2013 09:34 AM, Eleanor Dodson wrote:
Some of us resisted using an orthogonal format for coordinates, arguing that 
the output from a crystal structure should refer to crystal axes.
And since symmetry was a crystal property it was important that we could "see" 
it easily.  The PDB format won out,  but I still use coordconv a lot
to turn back the orthogonalised PDB style to fractional coordinates - to see if 
this heavy atom solution is the same as that one, given an origin shift, etc 
etc.
Eleanor

On 4 Jan 2013, at 20:44, Soisson, Stephen M wrote:



--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582




..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] 3D alignment of points (atoms)

[Bosch, Juergen]

can't you feed solve with those positions or Hyss or the Sharp interface ?
It's been too long ago when I faced this issue but I remember force-feeding one 
of the existing programs at the time with blank coordinates to figure it out - 
or was it mlphare ?

Too long ago probably 10 years by now.

Jürgen

On Dec 28, 2012, at 10:42 AM, Soisson, Stephen M wrote:

I did this exact thing ages ago to place a domain using just predicted SeMet 
positions using O's lsq commands.  Cumbersome syntax as I recall, but worked 
great.

Thanks,
Steve

On Dec 28, 2012, at 10:18 AM, "Jason Vertrees" 
mailto:jason.vertr...@schrodinger.com>> wrote:

Hi Dave,

If you're still searching for a quick way to do this, check out
PyMOL's Pair Fitting Wizard (Wizard > Pair Fitting; see Page 20
http://www.doe-mbi.ucla.edu/CHEM125/pymol_tutorial_060418.pdf). The
wizard is interactive and quick to use.

This can also be done from the command line or scripted using the
pair_fit command (http://www.pymolwiki.org/index.php/Pair_fit). We use
the Kabsch algorithm (with the appropriate corrections for reflection)
and a faster hand-rolled technique, using if I recall correctly Jacobi
rotations, to annihilate off-diagonal values.

Cheers,

-- Jason

--
Jason Vertrees, PhD
Director of Core Modeling Product Management
Schrödinger, Inc.

(e) jason.vertr...@schrodinger.com
(o) +1 (603) 374-7120


On Fri, Dec 28, 2012 at 5:53 AM, Tom Oldfield 
mailto:oldfi...@ebi.ac.uk>> wrote:
Hi

In you post you say you want to fit a  small number of points.  Note that
the
original algorithm of Kabsch has a number of maths pathalogical conditions
where points have symmetry or lie in a plane/line - this is common for
a small number of points (fitting residues or your example).

The maths for an update to this algorithm can be found here
Oldfield   St'Fun'Gen  (2002) 510-528   (appendix C) where cross terms
are used to generate the eigen vectors.  This algorithm is very stable
for fitting a small number of points and might be more suitable for
what you are trying to do.

If you want I can email you the code in C or maybe Java, though it
has rather a lot of other weighting schemes/masking used in the above
paper.

Regards
Tom


Lsqkabsch should do the trick.
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Dale Tronrud
Sent: Thursday, December 27, 2012 9:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3D alignment of points (atoms)


  If you just want the mathematics and are willing to roll your own
code, you can use the method of Wolfgang Kabsch.  I see this has been
enshrined in a Wikipedia page at

http://en.wikipedia.org/wiki/Kabsch_algorithm

This is what I've used when I've wanted to superimpose points where the
mapping between the points is defined.  If the points in your tetramer
aren't pathological, like lying in a common plane, you shouldn't have to
worry about SVD and can just perform the matrix inversion.

Dale Tronrud


On 12/27/12 11:16, Waugh, David (NIH/NCI) [E] wrote:

Greetings,

I have what seems like a relatively simple problem to solve, but have

not been able to do so using the software tools I know about. I have two
sets of 4 points in 3D space (atoms in PDB files). They represent
equivalent positions in two tetrameric proteins. I would like to align
these points in one PyMol or Coot file. I don't want a NEW set of points
representing the LSQ average of the two sets, which is what I get in
Coot's SuperPose. Instead I am looking for a way to "superimpose" one
atom from each set and then rotate one set for the best fit. I'm not an
intuitive expert on symmetry, but I think there is probably only one
best solution to this problem, right? I also need the atomic distances
to be on the same scale in the two sets of points.

Thanks for any help!

Dave Waugh

--
David S. Waugh, Ph.D.
Macromolecular Crystallography Laboratory Center for Cancer Research
National Cancer Institute Bldg. 538, Room 209A Frederick, MD
21702-1201
+1 (301) 846-1842
wau...@mail.nih.gov
http://mcl1.ncifcrf.gov/waugh.html
--
Notice:  This e-mail message, together with any attachments, contains
information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station,
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for the use of the individual or entity named on this message. If you are
not the intended recipient, and have received this message in error,
please notify us immediately by reply e-mail and then delete it from
your system.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Stre

Re: [ccp4bb] Today ...

[Bosch, Juergen]

Hi David,

on a computer I agree 20121221 is the way to go (but even then you could still 
do ls -lta), but on Eppendorf tubes that is precious real estate for other 
important numbers or letters hence the 12d21 :-)

And thanks for catching my double "J" this was just a test if somebody would 
actually read what I wrote :-)
And the corrected values were indeed right, aka following the simple rule I 
mentioned.

Jürgen

On Dec 21, 2012, at 9:15 AM, David Schuller wrote:

On neither case does an alphanumeric sort coincide with a chronological sort. 
The obvious solution is to petition to have the months renamed alphabetically.

On 12/21/12 03:23, Tom Murray-Rust wrote:
Hi Juergen,

Your scheme as printed has two J's - so January and July are indistinguishable! 
I would suggest the letters should instead be JFMAYULGSOND.

On 21 Dec 2012, at 01:52, "Bosch, Juergen" 
mailto:jubo...@jhsph.edu>> wrote:

May I introduce you to another fool proof way:
12d12 ...
J, F,M,A,Y,J,U,G,S,O,N,D for the months, simply first letter, but if taken move 
to the second letter etc.



--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu<mailto:schul...@cornell.edu>


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Today ...

[Bosch, Juergen]

May I introduce you to another fool proof way:
12d12
year-month-day
This is in particular useful in a mixed lab with american and europeans 
labeling Eppendorf tubes differently. The month has a defined letter, like this 
you know that your buffer was made on June 9th and not September 6th
J, F,M,A,Y,J,U,G,S,O,N,D for the months, simply first letter, but if taken move 
to the second letter etc.

I'm sure you guys know about the two Bohemian mathematicians who figured out 
that the Apocalypse as predicted by the Mayan calendar is due in 104 years 
because the alignment of events was done incorrectly.

Jürgen

..
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu


On Dec 20, 2012, at 12:17 PM, David Schuller wrote:

On 12/20/12 11:23, Edward A. Berry wrote:
No, No- in scientific circles we go from MSB on the left to LSB on the
right:
2012 12 20  (still a sort of palindrome).

This is the best method if you are going to incorporate the date into a
file name. That way alphanumeric and chronological searches come up the
same.

It's a pity it took us so long to figure this out, and the world is
ending tomorrow.

http://www.makemeacocktail.com/cocktail/7209/mayan-apocalypse/
Maya Apocalypse cocktail

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] archival memory?

[Bosch, Juergen]

Hey Dale,

you really should get your personal RAID with hot swappable discs, since you 
don't like Firewire, how about Thunderbolt and a Pegasus RAID with 6 bays ? If 
a drive fails you replace it with a new one.

By the way if anybody has a functional DAT4 tape drive, could I send you one to 
read out a tape with some data ? If so, then off list reply would be nice, 
thanks.

Jürgen

On Dec 12, 2012, at 5:22 PM, Dale Tronrud wrote:

   I don't believe there is a solution that does not involve active
management.  You can't write your data and pick up those media 25
years later and expect to get your data back -- not without some
heroic effort involving the construction of your own hardware.

   I have data from Brian Matthews' lab going back to the mid-1970's
and those data started life on 7-track mag tapes.  I've moved them
from there to 9-track 1600 bpi tapes, to 9-track 6250 bpi tapes, to
just about every density of Exabyte tape, to DVD, and most recently
to external magnetic hard drives (each with USB, Firewire, and eSATA
interfaces).  The hard drives are about five years old and so far
are holding up.  Last time I checked I could still read the 10 year
old DVD's.  I'm having real trouble reading Exabyte tapes.

   Write your data to some medium that you expect to last for at least
five years but anticipate that you will then have to move them to
something else.

   Instead of spending time working on the 100 year solution you should
spend your time annotating your data so that someone other than you
can figure out what it is.  Lack of annotation and editing is the
biggest problem with old data.

Dale Tronrud

P.S. If someone needs the intensities for heavy atom derivatives of
Thermolysin written in VENUS format, I'm your man.



On 12/12/2012 1:57 PM, Richard Gillilan wrote:
Better option? Certainly not TAPE or electromechanical disk drive. CD's and 
DVD's don't last nearly that long and James Holton has pointed out.

I suppose there might be a "cloud" solution where you rely upon data just 
floating around out there in cyberspace with a life of its own.

Richard

On Dec 12, 2012, at 4:41 PM, Dale Tronrud wrote:


  Good luck on your search in 100 years for a computer with a
USB port.  You will also need software that can read a FAT32
file system.

Dale "Glad I didn't buy a lot of disk drives with Firewire" Tronrud

On 12/12/2012 1:02 PM, Richard Gillilan wrote:
SanDisk advertises a "Memory Vault" disk for archival storage of photos that 
they claim will last 100 years.

(note: they do have a scheme for estimating lifetime of the memory, Arrhenius 
Equation ... interesting. Check it out: 
www.sandisk.com/products/usb/memory-vault/
 and click the Chronolock tab.).

Has anyone here looked into this or seen similar products?

Richard Gillilan
MacCHESS


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] To cryo or not to cryo... Mothe

[Bosch, Juergen]

I collected on 20% and 25% PEG4000 the trick though was small crystals (<100µm 
longest dimension) so the liquid can freeze (is that the right word, 
considering the 80+ thread we recently had) fast by plunging the mounted 
crystal into liquid nitrogen. A faint ice ring was still visible but didn't 
harm.
The previous suggestion with supplementing PEG400 into the mix is what I 
typically do. Keep your 30% PEG4000 and add either glycerol or PEG400, the 
latter is more safe as it is more similar.

Good luck,

Jürgen

On Nov 30, 2012, at 9:12 PM, Jackie Vitali wrote:

I would try your motherl liquor augmented with 10 per cent PEG 400 hoping for 
the best.  Better yet mount your crystal inside a capillary like old times & 
measure room temp data.  At least you have something for sure.  Finally I have 
heard (never did it) that if you do not have air on top of your dewar (like 
nitrogen atmosphere) you do not need cryoprotectant.

In my hands PEG4000 is not a cryoprotectant but adding 5 to 10 per cent PEG400 
does the job.

If I had an option I would go for room temp data in a home source.


Sent from my iPhone

On Nov 30, 2012, at 8:22 PM, Yuri Pompeu 
mailto:yuri.pom...@ufl.edu>> wrote:

Dear community,
I have what seems to be a pretty decent single crystal that grew from a screen 
set up 2 weeks ago.
I am trying to reproduce it but so far I have not succeeded. I am however 
afraid the crystal that did form will start to deteriorate. So this brings me 
to dilemma, I feel like I should try and mount this crystal and shoot it. But 
since I only have 1 sample, I do not want to mess this up...  I am inclined to 
try cryo conditions, but I am afraid the addition of a cryo such as glycerol 
could destroy the little guy.
The crystal formed in 30% PEG 4000, 0.1M NaCitrate pH5.6 and 0.2M NH4AcO, I 
wonder if this is a cryo condition already?
Any suggestions would be appreciated.

best,

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] OT: Has anyone experienced problems with Apple laptop battery expansion?

[Bosch, Juergen]

It probably had a functional Firewall under Linux that's why it survived :-)

Jürgen

P.S. note to myself, don't ever give George my laptop, as it might be 
mistreated - just for test purposes :-)

On Nov 20, 2012, at 12:57 PM, George M. Sheldrick wrote:

It is dual bootable Linux/Windows
George

On 11/20/2012 05:56 PM, William Scott wrote:
Presumably then it was running Linux?

On Nov 20, 2012, at 7:39 AM, "George M. Sheldrick" 
mailto:gshe...@shelx.uni-ac.gwdg.de>> wrote:

I have never understood why Macs are so popular, although in this part
of the world they are appreciably more expensive. My vintage 2005 Dell
laptop does not have the sex appeal of a MacBook, but it has survived a
fire (not its fault) as well as being under water (to put out the fire)
and then bounced down a stone staircase (my wife fortunately caught it
on the way down). Despite all my efforts to destroy it, it never needed
servicing or replacement parts and still works perfectly!

George
(PS I should add that I have no connection with or shares in Dell)


--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Collecting native data at an absorption edge

[Bosch, Juergen]

You'll see the zinc also at the Semet peak edge already check out Ethan's edge 
plot web server. If you used his-tag purification methods it would be wiser to 
collect passed the edge of either Ni or Co so you can distinguish them from Zn 
sites.

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://lupo.jhsph.edu

On Nov 20, 2012, at 9:26, "Alan Cheung"  wrote:

> Dear all - we have a synchrotron trip coming up and have crystals that 
> are likely to contain intrinsically bound zinc atoms.  The crystals 
> diffract to 4A-ish so far.  We're hoping to collect both native datasets 
> at higher dose, and phasing datasets at lower dose (on separate 
> crystals).  Can we do the lot at the zinc edge wavelength, or should we 
> be worried about radiation damage, and move away from the edge for the 
> native datasets?  And would a native dataset for refinement be 
> compromised by friedel pair differences collected at an edge?
> 
> Alan
> 
> 
> 
> 
> -- 
> Alan Cheung
> Gene Center
> Ludwig-Maximilians-University
> Feodor-Lynen-Str. 25
> 81377 Munich
> Germany
> Phone:  +49-89-2180-76845
> Fax:  +49-89-2180-76999
> E-mail: che...@lmb.uni-muenchen.de

Re: [ccp4bb] OT: Has anyone experienced problems with Apple laptop battery expansion?

[Bosch, Juergen]

Bill I think that's crap.
I had issues on a 2005 MacBook Pro with inflating battery and it was replaced 
(after about 6 months). There were troubles with those batteries and impurities 
but mine still had apple care at that time and the batteries were exchangeable. 
I have not heard of the build in batteries to have problems but yours sure did. 
Send Tim Cook an email with the picture. This should not have happened and also 
keeping the power cord on leading to this problem should not have happened. For 
what did they introduce the trickling charging ? If you can't leave the coord 
plugged in how many nice wooden US households gave caught fire due to Apple 
products ?

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://lupo.jhsph.edu

On Nov 17, 2012, at 16:28, "William G. Scott"  wrote:

> Hi folks:
> 
> I'm trying to get a sense for how frequently this sort of thing occurs:
> 
> 
> 
> That was a macbook air that served me well for four years, but then 
> self-destructed. (I took it to the Apple store.  They generously offered to 
> repair it for $800 or to sell me a new one, and suggested this was normal if 
> you leave the power cord attached after the battery charges, even while 
> giving a lecture or seminar.)  It strikes me as a bit dangerous.
> 
> --Bill Scott
> 
> 
> 
> 
> 
> William G. Scott
> Professor
> Department of Chemistry and Biochemistry
> and The Center for the Molecular Biology of RNA
> 228 Sinsheimer Laboratories
> University of California at Santa Cruz
> Santa Cruz, California 95064
> USA
> 
> 
> 

Re: [ccp4bb] Off topic: Selecting atoms within a given distance from a target atom

[Bosch, Juergen]

Hi Pavel,

does this also work for symmetry related atoms ?

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://lupo.jhsph.edu

On Nov 17, 2012, at 14:26, "Pavel Afonine" 
mailto:pafon...@gmail.com>> wrote:

Hi Rex,

as easy as:

phenix.pdb_atom_selection model.pdb "within(3, chain L and resseq 9 and name 
CA)" --write-pdb-file=cut.pdb

which in the above example selects all atoms within 3 A from CA atom in chain A 
of residue number 9, and writes them into cut.pdb file.

Pavel

On Sat, Nov 17, 2012 at 12:04 PM, Rex Palmer 
mailto:rex.pal...@btinternet.com>> wrote:
I would like to specify a target atom in a pdb file and then isolate all atoms 
within a given distance of the target. The selected atoms are then to be placed 
in a new pdb file.
Any suggestions please.

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Re: [ccp4bb] vitrification vs freezing

[Bosch, Juergen]

HI Tim,

you should know better. German is the most precise language, hence all those 
old German *gosh* books (for the younger readers of this board, there was a 
time before pdf and Nook readers) for organic chemistry etc. from the 19th 
century and older (Beilstein, Angewandte ...). And why was that the case ? 
Because we love-to-connect-three-or-five-words in one describing all of the 
above :-)

how about "data was collected at -180˚C (93.15K)", fairly precise the reader 
can think if it's frozen or vitrified and the writer couldn't care less.

flash-annealing - any takers on that one ?
Transition from hexagonal ice to vitrified glass - or just magic ?

Jürgen


On Nov 16, 2012, at 4:54 AM, Tim Gruene wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi James,

I once heard that in (European) law French is the language of choice
because it were the most precise one (which I find easy to believe).
Maybe we should try and convince journals to only accept articles
written in French - not sure, this will improve their quality, though,
comparing my level of French with my level of English ;-)

Lovely discussion,
Tim

On 11/15/2012 09:15 PM, James Stroud wrote:
On Nov 15, 2012, at 10:59 AM, Tim Gruene wrote:
I have heard this discussion before and reminds me of people
claiming strawberries were nuts - which botanically may be
correct, but would still not make me complain about strawberries
in a fruit cake I ordered at a restaurant.

My Pengiun English Dictionary states (amongst other
explanations) freeze: "to make extremely cold",


Tim's comment strikes at the heart of the problem.

I think the scientific community should decide a few points.

1. What is the approved language and dialect for science? 2. Within
this dialect, what should be the authoritative dictionary? 3. Will
we allow use of definitions that are not the primary definition
(second, third, fourth). 4. Will we allow the use of homonyms? 5.
If not, which homonyms should prevail?

These are all very important questions if we completely disregard
context in writing.

James


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

GPG Key ID = A46BEE1A

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..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] off topic: oligomer detection

[Bosch, Juergen]

What if your mild urea condition unfolds your protein from a globular to a 
stretched elongated form ? You probably have done CD already to verify that 
this is not the case.
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://lupo.jhsph.edu

On Nov 14, 2012, at 2:57, "Careina Edgooms" 
mailto:careinaedgo...@yahoo.com>> wrote:

Anyone have any advice on how to detect whether my protein is forming 
oligomers? It is monomeric in the native state but I have reason to believe 
that it may be oligomerising in mild concentrations of urea (intermediate 
state).
I have tried cross linking and BN PAGE and they are inconclusive. SE-HPLC does 
not work because at the concentrations required to produce a signal, this 
intermediate species aggregates.
I do not have access to an ultracentrifuge. The dynamic light scattering 
equipment that is available to me is really a poor instrument which will not be 
sensitive enough to pick up changes in size (we are looking at about 5nm for 
the monomer and anything from 8nm for the oligomer). The only other option I 
can think of is SAXS. I will only be able to use that equipment in the middle 
of next year.
I'm wondering if there is anything else, any other technique or idea that I 
have not thought about that I could try? I really just would like to show that 
the stoichiometry of the intermediate species is a multiple of the native state.
If anyone has any suggestions, that would be great.
Thanks
Careina.

Re: [ccp4bb] Apologies

[Bosch, Juergen]

Hi Matthias,

I have not read your attached MS however Figure 2 is very good. Congrats to the 
first author for a very captive and engaging view of your protein.

Jürgen

On Nov 12, 2012, at 4:24 AM, Matthias Wilmanns wrote:

I am apologizing for sending an unintended email to CCP4 via CC.

Matthias, EMBL Hamburg

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] usefulness of cacodylate?

[Bosch, Juergen]

Well originally we got these in cacodylate and not much else would diffract.
http://www.pdb.org/pdb/explore/materialsAndMethods.do?structureId=2EPH
But nowadays I have another conditions, which does not require cacodylate and 
works well with Hepes.

Jürgen

On Nov 9, 2012, at 7:26 AM, Frank von Delft wrote:

> Hi all -
> 
> Anybody know
> a) how hazardous is cacodylate?
> b) does it really matter for crystallization screens?
> 
> It seems by far the most hazardous component of the standard screens;  
> this 2011 paper seems to think so (bizarrely, I can't access it from 
> Oxford):
> http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract
> 
> and this is site says lethal dose is 0.5-5g/kg:
> http://cameochemicals.noaa.gov/chemical/4468
> meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone 
> should check my maths...)  [Coarse screens come mixed 2ml per condition.]
> 
> 
> Has anybody done careful experiments that showed it really mattered for 
> a given crystal -- or even an entire screen?
> 
> So I'm inclined to toss it out entirely rather than make crystallization 
> screening a "hazardous activity".  (We're being subjected to a safety 
> review.)
> 
> 
> Thoughts welcome.
> phx

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu