Re: [ccp4bb] One little clash

[James Stroud]

The hydroxyls were on the wrong carbons in the previous picture I sent. These are correct.JamesOn Jul 11, 2012, at 1:37 PM, Lukacs, Christine wrote:Hi all- I have a protein that crystallizes in I422, and diffracts well, between 1.3-1.7A.  Beautiful density, slightly higher final R-factors than you might expect at this resolution (low to mid 20s).  The density is all beautiful, except that I have this one little clash, between a few atoms from a tyrosine and its symmetry mate.  In this picture I have it modeled as an Alanine and you can see the two tyrosine rings interlocking; and there is clearly no alternate conformation.  Since it is not near my site of interest, I have been pretty much ignoring it, going through refinement with it as an alanine, then changing it at the very end to a tyrosine and just minimizing B-s, no positional.  Now that I plan to publish a bunch of these, I should probably figure out what is really going on.  Any insights? ThanksChristineChristine Lukacs RocheThis message is intended for the use of the named recipient(s) only and may contain confidential and/or proprietary information. If you are not the intended recipient, please contact the sender and delete this message. Any unauthorized use of the information contained in this message is prohibited. 

Re: [ccp4bb] One little clash

[James Stroud]

It looks like dityrosine, usually caused by radiation damage (I think UV is the usual culprit).http://www.nugowiki.org/images/thumb/c/ca/HMDB06045.png/220px-HMDB06045.pngJamesOn Jul 11, 2012, at 1:37 PM, Lukacs, Christine wrote:Hi all- I have a protein that crystallizes in I422, and diffracts well, between 1.3-1.7A.  Beautiful density, slightly higher final R-factors than you might expect at this resolution (low to mid 20s).  The density is all beautiful, except that I have this one little clash, between a few atoms from a tyrosine and its symmetry mate.  In this picture I have it modeled as an Alanine and you can see the two tyrosine rings interlocking; and there is clearly no alternate conformation.  Since it is not near my site of interest, I have been pretty much ignoring it, going through refinement with it as an alanine, then changing it at the very end to a tyrosine and just minimizing B-s, no positional.  Now that I plan to publish a bunch of these, I should probably figure out what is really going on.  Any insights? ThanksChristineChristine Lukacs RocheThis message is intended for the use of the named recipient(s) only and may contain confidential and/or proprietary information. If you are not the intended recipient, please contact the sender and delete this message. Any unauthorized use of the information contained in this message is prohibited. 

Re: [ccp4bb] Crystal Optimization

[Muhammed bashir Khan]

Dear All,

Thanks alot for your valuable suggestion.I hope I will find out the
solution now. As far as to giveup is out of question

Thanks once agin

Regards;

Bashir


On Wed, July 11, 2012 05:25, Tuhin Bhowmick wrote:
> Dear Muhammad,
>
> I had a similar case, and the crystals could indeed be optimized. A few
> things to check first,
>
> 1) How long does it take for the needles to appear? Sometimes, if the
> protein is degraded/ cleaved over time, a small population (possibly a
> fragment of the whole
>protein) from the heterogeneous mix can give similar crystals. Like
> Bryan had suggested, it is also very useful to check the mol wt. of the
> crystallized species through
>mass spect/ native page. But do make sure to give the crystals serial
> washes, so the test accounts for crystallizexd species, not the ones from
> surrounding condition.
>
> 2) If the crystals indeed contain the protein of interest, they can be
> used
> for various seeding methods. I've got results from both streak and micro
> seeding.
>
> Best,
>
> Tuhin.
>
> Tuhin Bhowmick
>
> Department of Physics
>
> Indian Institute of Science
>
> Bangalore: 560012
> Email: tuhin.i...@gmail.com
>
> On Wed, Jul 11, 2012 at 12:34 AM, Bernhard Rupp (Hofkristallrat a.D.) <
> hofkristall...@gmail.com> wrote:
>
>> > Always give up.
>>
>> ** **
>>
>> ...definitely not the kind of guy I want to sit up front in an
>> airliner…**
>> **
>>
>> ** **
>>
>> Best regards, BR
>>
>> -
>>
>> Bernhard Rupp, ATP-B737, CFII-MEI
>>
>> Vienna Air International
>>
>> Professional Aviation Services
>>
>> 001 (925) 209-7429
>>
>> +43 (676) 571-0536
>>
>> b...@vienna-air.com
>>
>> b...@ruppweb.org
>>
>> http://www.vienna-air.com/   
>>
>> -
>>
>> It is not your aptitude but your attitude
>>
>> that determines your altitude. (or your crystals)
>>
>> -
>>
>> ** **
>>
>> ** **
>>
>> ** **
>>
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
>> yybbll
>>
>> Sent: Tuesday, July 10, 2012 11:58 AM
>>
>> To: CCP4BB@JISCMAIL.AC.UK
>>
>> Subject: Re: [ccp4bb] Crystal Optimization
>>
>> ** **
>>
>> Hi, 
>>
>>  
>>
>> In my experience, it is very very very difficult to optimize this needle
>> like crystal. Always give up.
>>
>>  
>>
>> Good luck!
>>
>>  
>>
>>  
>>
>> Dear All;
>>
>> Could somebody give a nice suggestion how the following type crystal
>> could
>> 
>>
>> be optimized, I almost tried everything.
>>
>> Crystal Image is attached
>>
>> Crystal condition: 20% w/v PEG3350 and 200mM NaCl.
>>
>> Thanks in advance
>>
>> Bashir
>>
>> -- 
>>
>> Muhammad Bashir Khan
>>
>> **
>>
>> Structural Genome Consortium (SGC). University of Toronto
>>
>> Toronto, Canada
>>
>


-- 
Muhammad Bashir Khan
**
Department for Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Austria

Phone: +43(1)427752224
Fax: +43(1)42779522

[ccp4bb] New Opening - Technical Key Account Manager at Microlytic

[Melanie Adams-Cioaba]

Hello all,

We are excited to open a new position at our company.  Please see the job
posting below.  Both formal and informal inquires may be submitted to
j...@microlytic.com.

Best wishes,
Melanie


*Technical Key Account Manager*

* *

*Position Summary:*

Your primary focus will be the commercialization of Microlytic products
into the workflow of a set of key accounts as well as other accounts. Your
primary responsibility is to drive sales growth while maintaining existing
business within the territory, in order to meet or exceed revenue goals.
The position will involve significant travel to customer sites, primarily
in North America, but from time to time worldwide, as the business may
require, to reach our annual sales goals. Your day-to-day activities will
focus on on-site meetings with customers, solving their technical problems
and facilitating the integration of Microlytic products into their
workflow. The ideal candidate will reside in Boston, San Francisco or San
Diego.  Compensation potential is in excess of $100,000.

* *

*ESSENTIAL DUTIES AND RESPONSIBILITIES:** *

• Present product information and discuss crystallography research
with customers and prospective customers in your assigned territory.

• Meet or exceed revenue goals and other goals as assigned.

• Leverage your technical expertise to meet or exceed your sales
targets.

• Increase our competitive advantage and enhance customer
satisfaction

• Build effective business and collaboration relationships with
customers.

• Prospecting to develop new accounts and arrange sales meetings.

• Product demonstrations in the field.

• Develop tactical plans to maximize revenues.

• Complete and submit sales reports and documentation in a timely
and efficient manner.

• Provide regular reports on customer inquiries, and communicate
successes, failures, best practices etc to improve the overall product
offering.

• Present technical seminars in person, via telephone or web
conference

• Gather and report customer and technical information

• Represent Microlytic at tradeshows/exhibitions and other
“on-site” events in order to promote crystallography products and services,
receive feedback, network with customers and prospective customers.

• Monitor competitive landscape and provide analysis of key
findings to senior management.

• This work will entail significant travel to customer sites.



*Qualifications and experience:*

• Minimum B.S. degree in life sciences, Ph.D. or M.S. preferred.

• Minimum of 3 years lab experience with proven knowledge and
understanding of structural biology, preferably within crystallography.

• Technical competency to interpret customer’s needs and identify
solutions; to stay current in technical knowledge; to troubleshoot and to
provide information back to customers in a helpful, courteous, positive and
professional manner.

• Sales experience, and prior success with building and sustaining
high growth of sales to pharmaceutical and biotechnology companies, as well
as academic and research institutes and federal research agencies, is
preferred.

• Possess a well developed network of customer contacts within the
life sciences industry.

• Ability to navigate through targeted organizations to understand
the internal dynamics and the decision-making processes of those companies,
and the ability to acquire and learn the information to understand
customer’s needs and requirements.

• Excellent oral and written communication skills.

• Experience in presenting technical materials in written and
verbal form is critical.

• Strong commitment to customer service and satisfaction.

• Ability to effectively work on and manage many priorities at one
time.

• A clear and persuasive communication style, with the ability to
gain trust and respect and to command attention both externally and
internally.

• A supportive and approachable team player, who is able to
challenge in a constructive manner, yet not hesitate to show independent
and creative thinking, and be able to compromise in order to accomplish the
objective.

• Must be self-reliant and a self-starter, with an entrepreneurial
spirit, who is results oriented, has a sense of urgency, and has a
competitive desire to succeed.



*About Microlytic*

Founded in 2006, Microlytic is an innovator and commercial provider of
consumable laboratory products for the structural biology marketplace. Our
mission is to provide a portfolio of innovative products that simplify the
workflow, increase productivity and offer an attractive economic value
proposition to our customers. For more information about Microlytic, please
visit http://www.microlytic.com.

Re: [ccp4bb] FE-Sulfur proteins

[Bosch, Juergen]

Have you looked at a UV-vis scan, there are some characteristic bumps you 
should see depending on the state of your FeS cluster, plus you can monitor if 
oxidation occurs over time. If I remember it correctly 322 nm is where you want 
to look at.

Jürgen

On Jul 11, 2012, at 6:22 AM, Jan Rashid Umar wrote:

Dear All,

I will be grateful to your suggestions about Iron-Sulfur cluster protein 
purification. My colleague has some problems with the purification and it seems 
that the iron-sulfur cluster might be deformed, and protein is aggregated. The 
purification is done under aerobic (normal condition), and is there some method 
to reintegrate iron sulfur cluster back into the protein molecule under these 
conditions. Can anybody suggest some literature, protocol or something that can 
improve the protein aggregation?

I look forward to hearing from you.

Best wishes,

Jan

..
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] FE-Sulfur proteins

[Colbert, Christopher]

Hi Jan,

How is the Fe-S cluster deformed?  What is the evidence?  Has your colleague 
done Fe and S analysis?  There is a lot of missing information to this question 
that prevents a much more thorough answer.  Is the protein membrane associated? 
Part of a larger complex?  Besides the Fe-S cluster there are a number of other 
things that can be an issue.  However,  many Fe-S clusters are sensitive to 
aerobic conditions.  If your colleague's cluster is structural, then when it 
degrades under aerobic conditions this can lead to protein aggregation or 
instability.  How well does the protein express?  It is also possible during 
overexpression to out pace the Isc machinery of the E. coli cell and thus not 
have full integration of your Fe-S cluster, which again could lead to 
aggregation.  I think the solutions to either of these scenarios are obvious 
and may require a collaborator to really determine the best way to pursue this 
protein purification.  As has been mentioned an exogenous Isc operon plasmid 
can be used to mitigate the effects of the later situation, but is not the only 
solution.

Good luck,

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

From: Jan Rashid Umar mailto:jan...@googlemail.com>>
Reply-To: Jan Rashid Umar mailto:jan...@googlemail.com>>
To: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] FE-Sulfur proteins

Dear All,

I will be grateful to your suggestions about Iron-Sulfur cluster protein 
purification. My colleague has some problems with the purification and it seems 
that the iron-sulfur cluster might be deformed, and protein is aggregated. The 
purification is done under aerobic (normal condition), and is there some method 
to reintegrate iron sulfur cluster back into the protein molecule under these 
conditions. Can anybody suggest some literature, protocol or something that can 
improve the protein aggregation?

I look forward to hearing from you.

Best wishes,

Jan

Re: [ccp4bb] Mercury phenylglyoxal

[Fischmann, Thierry]

http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152___13721198_-1
 

Is this what you're looking for?

Thierry

> Hello all,
>
> Could someone please advise me on where to purchase Mercury Phenylglyoxal
> from?
>
> Many thanks,
> Vijay
>
> 
> Vijay S. Reddy, Ph.D.
> Associate Professor
> Department of Molecular Biology, TPC6
> The Scripps Research Institute,
> 10550 North Torrey Pines Road,
> La Jolla, CA 92037
>
> E-mail: red...@scripps.edu
> WWW:http://www.scripps.edu/~reddyv
> VIPERdb:http://viperdb.scripps.edu
>
> Phone:(858) 784-8191
> FAX:(858) 784-8896
> 
Notice:  This e-mail message, together with any attachments, contains
information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station,
New Jersey, USA 08889), and/or its affiliates Direct contact information
for affiliates is available at 
http://www.merck.com/contact/contacts.html) that may be confidential,
proprietary copyrighted and/or legally privileged. It is intended solely
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.

Re: [ccp4bb] Disulphide bonds and closed conformation

[Dr. Lorenzo Finci]

Jan, 
I agree that the DTNB assay (Ellman's Reagent) can be used to quantify exposed 
Cysteines. Alternatively, you could do site directed mutagenesis and mutate 
significant amino acids responsible for the conformational change from the open 
to the closed state, with hopes to lock the protein in the closed conformation. 
However, this may prove to be tricky. If the protein is indeed an enzyme, you 
can also attempt to crystallize the protein with a transition state analog 
inhibitor, thus inhibiting turnover and locking the protein in the closed 
conformation
Good luck!lorenzo 

Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard 
Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe 
College of Life SciencesBeijing, China


Date: Wed, 11 Jul 2012 11:12:17 -0400
From: kellydaugh...@gmail.com
Subject: Re: [ccp4bb] Disulphide bonds and closed conformation
To: CCP4BB@JISCMAIL.AC.UK

Jan,If the Cys residues are accessible, you could try DTNB to quantify the 
number of Cys, thus determining if they are reduced or bridged.

http://en.wikipedia.org/wiki/Ellman's_reagent


Kelly***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building


303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***



On Wed, Jul 11, 2012 at 11:08 AM, Jan Rashid Umar  wrote:


Dear all,
I
 am working on a protein where I have to stabilize the closed 
conformation of the protein using disulphide bond. The strategy to 
design the cysteine mutants is based on the molecular dynamic 
simulations, and accordingly the residues were chosen. The ultimate goal
 is to trap the ligand in closed conformation of protein and crystallize
 it. I am facing few issues: Is there some reliable assay that can check
 the formation of disulphide bonds in protein.  Additionally, does 
anybody knows another method(s) that can be used to trap a closed 
conformation. I look forward for your suggestions and discussions on 
this issue. 
Thanks very much!

Jan


  

Re: [ccp4bb] Disulphide bonds and closed conformation

[Allan Pang]

Ellman's assay may help you.

http://en.wikipedia.org/wiki/Ellman's_reagent
http://www.piercenet.com/instructions/2160311.pdf

Allan

Quoting Jan Rashid Umar :


Dear all,

I am working on a protein where I have to stabilize the closed conformation
of the protein using disulphide bond. The strategy to design the cysteine
mutants is based on the molecular dynamic simulations, and accordingly the
residues were chosen. The ultimate goal is to trap the ligand in closed
conformation of protein and crystallize it. I am facing few issues: Is
there some reliable assay that can check the formation of disulphide bonds
in protein.  Additionally, does anybody knows another method(s) that can be
used to trap a closed conformation. I look forward for your suggestions and
discussions on this issue.

Thanks very much!

Jan





--
Allan Pang

PhD Student

G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS

Phone number: 02078828480

Twitter: @xerophytes

Re: [ccp4bb] Disulphide bonds and closed conformation

[Kelly Daughtry]

Jan,
If the Cys residues are accessible, you could try DTNB to quantify the
number of Cys, thus determining if they are reduced or bridged.


http://en.wikipedia.org/wiki/Ellman's_reagent

Kelly

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Wed, Jul 11, 2012 at 11:08 AM, Jan Rashid Umar wrote:

> Dear all,
>
> I am working on a protein where I have to stabilize the closed
> conformation of the protein using disulphide bond. The strategy to design
> the cysteine mutants is based on the molecular dynamic simulations, and
> accordingly the residues were chosen. The ultimate goal is to trap the
> ligand in closed conformation of protein and crystallize it. I am facing
> few issues: Is there some reliable assay that can check the formation of
> disulphide bonds in protein.  Additionally, does anybody knows another
> method(s) that can be used to trap a closed conformation. I look forward
> for your suggestions and discussions on this issue.
>
> Thanks very much!
>
> Jan
>

[ccp4bb] Disulphide bonds and closed conformation

[Jan Rashid Umar]

Dear all,

I am working on a protein where I have to stabilize the closed conformation
of the protein using disulphide bond. The strategy to design the cysteine
mutants is based on the molecular dynamic simulations, and accordingly the
residues were chosen. The ultimate goal is to trap the ligand in closed
conformation of protein and crystallize it. I am facing few issues: Is
there some reliable assay that can check the formation of disulphide bonds
in protein.  Additionally, does anybody knows another method(s) that can be
used to trap a closed conformation. I look forward for your suggestions and
discussions on this issue.

Thanks very much!

Jan

Re: [ccp4bb] FE-Sulfur proteins

[Kelly Daughtry]

I *believe* that the ISC operon is utilized in E. coli for all FeS
clusters, regardless if Rieske or not.
I know that when deleted, activity of FeS containing proteins (succinate
dehydrogenase and Glutamate synthase) greatly decreases. See:
http://www.ncbi.nlm.nih.gov/pubmed/11432781

Thus it *should* work with other FeS containing enzymes.

Kelly


***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Wed, Jul 11, 2012 at 9:43 AM, Edward A. Berry  wrote:

> Good to know about this ISC operon!
>
> Do you know if it is specific for Rieske-type His2/Cys2 Fe2S2 clusters,
> or for FeS clusters in general?
> Thus wild-type E. coli is already making three types of ISC clusters
> for succinate dehydrogenase or fumarate reductase (although some help
> might be needed for overexpression) but I don't know if it has
> any Rieske-type clusters.
>
>
> Kelly Daughtry wrote:
>
>> Jan,
>> Do you express the protein with the /E. coli isc/ iron-sulfur cluster
>> synthetic operon?
>>
>> I found great success, see:
>>
>> Daughtry, KD et. al. JACS 2012
>> http://pubs.acs.org/doi/full/**10.1021/ja2111898
>>
>> - Kelly Daughtry
>>
>> *
>> Kelly Daughtry, Ph.D.
>> Post-Doctoral Fellow, Raetz Lab
>> Biochemistry Department
>> Duke University
>> Alex H. Sands, Jr. Building
>> 303 Research Drive
>> RM 250
>> Durham, NC 27710
>> P: 919-684-5178
>> *
>>
>>
>> On Wed, Jul 11, 2012 at 6:22 AM, Jan Rashid Umar > **> wrote:
>>
>> Dear All,
>>
>> I will be grateful to your suggestions about Iron-Sulfur cluster
>> protein purification.
>> My colleague has some problems with the purification and it seems
>> that the iron-sulfur
>> cluster might be deformed, and protein is aggregated. The
>> purification is done under
>> aerobic (normal condition), and is there some method to reintegrate
>> iron sulfur
>> cluster back into the protein molecule under these conditions. Can
>> anybody suggest
>> some literature, protocol or something that can improve the protein
>> aggregation?
>>
>> I look forward to hearing from you.
>>
>> Best wishes,
>>
>> Jan
>>
>>
>>
>

Re: [ccp4bb] Mercury phenylglyoxal

[Vijay Reddy]

Okay, that settles it.

Thank you David and Pat for sharing the knowledge and wisdom.

Kind regards,
Vijay

On Jul 11, 2012, at 6:43 AM, Patrick Loll wrote:

I second the mythical conclusion. We also tried to make it and  
failed 10-15 yrs ago (we came up with the di-iodo compound, if I  
recall). Many of these organomercurials are really tough to handle;  
I suspect that even if we had succeeded in making the desired  
compound, it would have been as soluble as a stone.


Pat

On 11 Jul 2012, at 9:18 AM, David Briggs wrote:


Hi Vijay,

Not an answer to your question, but I tried and failed to source any
about 10 years ago. I had a chemist friend try to make some following
the a protocol we found on the web - which didn't work.

The consensus on the ccp4bb back then was that it was a bit of
mythical beast, and I never progressed beyond this.
http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00958.html

We made some Iodo-phenylglyoxal though... never got it to work (as a
derivative) in action.

Cheers,

Dave


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


On 11 July 2012 13:37, Vijay Reddy  wrote:

Hello all,

Could someone please advise me on where to purchase Mercury  
Phenylglyoxal

from?

Many thanks,
Vijay


Vijay S. Reddy, Ph.D.
Associate Professor
Department of Molecular Biology, TPC6
The Scripps Research Institute,
10550 North Torrey Pines Road,
La Jolla, CA 92037

E-mail: red...@scripps.edu
WWW:http://www.scripps.edu/~reddyv
VIPERdb:http://viperdb.scripps.edu

Phone:(858) 784-8191
FAX:(858) 784-8896




Vijay S. Reddy, Ph.D.
Associate Professor
Department of Molecular Biology, TPC6
The Scripps Research Institute,
10550 North Torrey Pines Road,
La Jolla, CA 92037

E-mail: red...@scripps.edu
WWW:http://www.scripps.edu/~reddyv
VIPERdb:http://viperdb.scripps.edu

Phone:(858) 784-8191
FAX:(858) 784-8896

Re: [ccp4bb] Mercury phenylglyoxal

[Patrick Loll]

I second the mythical conclusion. We also tried to make it and failed 10-15 yrs 
ago (we came up with the di-iodo compound, if I recall). Many of these 
organomercurials are really tough to handle; I suspect that even if we had 
succeeded in making the desired compound, it would have been as soluble as a 
stone.

Pat

On 11 Jul 2012, at 9:18 AM, David Briggs wrote:

> Hi Vijay,
> 
> Not an answer to your question, but I tried and failed to source any
> about 10 years ago. I had a chemist friend try to make some following
> the a protocol we found on the web - which didn't work.
> 
> The consensus on the ccp4bb back then was that it was a bit of
> mythical beast, and I never progressed beyond this.
> http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00958.html
> 
> We made some Iodo-phenylglyoxal though... never got it to work (as a
> derivative) in action.
> 
> Cheers,
> 
> Dave
> 
> 
> David C. Briggs PhD
> http://about.me/david_briggs
> 
> 
> On 11 July 2012 13:37, Vijay Reddy  wrote:
>> Hello all,
>> 
>> Could someone please advise me on where to purchase Mercury Phenylglyoxal
>> from?
>> 
>> Many thanks,
>> Vijay
>> 
>> 
>> Vijay S. Reddy, Ph.D.
>> Associate Professor
>> Department of Molecular Biology, TPC6
>> The Scripps Research Institute,
>> 10550 North Torrey Pines Road,
>> La Jolla, CA 92037
>> 
>> E-mail: red...@scripps.edu
>> WWW:http://www.scripps.edu/~reddyv
>> VIPERdb:http://viperdb.scripps.edu
>> 
>> Phone:(858) 784-8191
>> FAX:(858) 784-8896
>> 

Re: [ccp4bb] FE-Sulfur proteins

[Edward A. Berry]

Good to know about this ISC operon!

Do you know if it is specific for Rieske-type His2/Cys2 Fe2S2 clusters,
or for FeS clusters in general?
Thus wild-type E. coli is already making three types of ISC clusters
for succinate dehydrogenase or fumarate reductase (although some help
might be needed for overexpression) but I don't know if it has
any Rieske-type clusters.


Kelly Daughtry wrote:

Jan,
Do you express the protein with the /E. coli isc/ iron-sulfur cluster synthetic 
operon?
I found great success, see:

Daughtry, KD et. al. JACS 2012
http://pubs.acs.org/doi/full/10.1021/ja2111898

- Kelly Daughtry

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Wed, Jul 11, 2012 at 6:22 AM, Jan Rashid Umar mailto:jan...@googlemail.com>> wrote:

Dear All,

I will be grateful to your suggestions about Iron-Sulfur cluster protein 
purification.
My colleague has some problems with the purification and it seems that the 
iron-sulfur
cluster might be deformed, and protein is aggregated. The purification is 
done under
aerobic (normal condition), and is there some method to reintegrate iron 
sulfur
cluster back into the protein molecule under these conditions. Can anybody 
suggest
some literature, protocol or something that can improve the protein 
aggregation?

I look forward to hearing from you.

Best wishes,

Jan

Re: [ccp4bb] off topic detection of oligomerisation

[Carlos Kikuti]

Urea?!?!? My arm hair went up.

Anyways, DLS (Dynamic Light Scattering) might help. Hard to imagine something 
quicker and easier, provided that someone next to you already has a DLS machine.


(sorry for the late answer)

Carlos



Em 26/06/2012, às 15:10, Brad Bennett escreveu:

> Native PAGE (i.e. BN-PAGE), light scattering (i.e. MALLS)
> 
> Not quick and easy but could work: AUC (i.e. a sedimentation equilibrium 
> experiment)
> 
> -Brad
> 
> On Tue, Jun 26, 2012 at 9:01 AM, Careina Edgooms  
> wrote:
> Dear ccp4 bulletin board
> 
> I apologise for off topic question. I wonder if anybody knows of a good 
> method to detect oligomerisation? 
> I suspect an equilibrium intermediate is forming oligomers based on 
> tryptophan fluorescence showing an exposed tryptophan becoming buried in a 
> hydrophobic region. I would like to perform some experiments to prove this. 
> Cross linking is not working for me and neither is SEC HPLC due to technical 
> issues with the urea I think.
> Are there any other quick and easy techniques that any of you can think of 
> off the cuff? Shifts in equilibrium unfolding curves done at different 
> concentrations will probably show something because of the law of mass action 
> but this is a big job needing many replicates so I wonder if there are any 
> other simple methods available?
> 
> Thanks
> Careina
> 

Re: [ccp4bb] Mercury phenylglyoxal

[David Briggs]

Hi Vijay,

Not an answer to your question, but I tried and failed to source any
about 10 years ago. I had a chemist friend try to make some following
the a protocol we found on the web - which didn't work.

The consensus on the ccp4bb back then was that it was a bit of
mythical beast, and I never progressed beyond this.
http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00958.html

We made some Iodo-phenylglyoxal though... never got it to work (as a
derivative) in action.

Cheers,

Dave


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


On 11 July 2012 13:37, Vijay Reddy  wrote:
> Hello all,
>
> Could someone please advise me on where to purchase Mercury Phenylglyoxal
> from?
>
> Many thanks,
> Vijay
>
> 
> Vijay S. Reddy, Ph.D.
> Associate Professor
> Department of Molecular Biology, TPC6
> The Scripps Research Institute,
> 10550 North Torrey Pines Road,
> La Jolla, CA 92037
>
> E-mail: red...@scripps.edu
> WWW:http://www.scripps.edu/~reddyv
> VIPERdb:http://viperdb.scripps.edu
>
> Phone:(858) 784-8191
> FAX:(858) 784-8896
> 

Re: [ccp4bb] sulphur bridge

[Edwin Pozharski]

If you need reducing agent, the best choice is probably TCEP.  If you 
are to choose between the BME and DTT, I'd recommend DTT, since BME 
tends to modify cysteines (sometimes in active site which may be quite 
annoying, although post-crystallization DTT soaks can remove some of 
these).  On the other hand, I know of at least one case whereby CYS-BME 
modification has helped crystallization by mediating a crystal contact.


In summary, every protein is different and if you have capacity to try 
BME, DTT and TCEP, try all three.  And no reducing agent as well.



On 07/11/2012 04:50 AM, amro selem wrote:

Hallo every one,
i am working on one enzyme who has sulphur bridge , is using 2 
mercaptoethnol or DTT  during handling this protein for 
crystallography is not desirable .

best regards
Amr

Re: [ccp4bb] FE-Sulfur proteins

[Kelly Daughtry]

Jan,
Do you express the protein with the *E. coli isc* iron-sulfur cluster synthetic
operon?
I found great success, see:

Daughtry, KD et. al. JACS 2012
http://pubs.acs.org/doi/full/10.1021/ja2111898

- Kelly Daughtry

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Wed, Jul 11, 2012 at 6:22 AM, Jan Rashid Umar wrote:

> Dear All,
>
> I will be grateful to your suggestions about Iron-Sulfur cluster protein
> purification. My colleague has some problems with the purification and it
> seems that the iron-sulfur cluster might be deformed, and protein is
> aggregated. The purification is done under aerobic (normal condition), and
> is there some method to reintegrate iron sulfur cluster back into the
> protein molecule under these conditions. Can anybody suggest some
> literature, protocol or something that can improve the protein aggregation?
>
> I look forward to hearing from you.
>
> Best wishes,
>
> Jan
>

[ccp4bb] Mercury phenylglyoxal

[Vijay Reddy]

Hello all,

Could someone please advise me on where to purchase Mercury  
Phenylglyoxal from?


Many thanks,
Vijay


Vijay S. Reddy, Ph.D.
Associate Professor
Department of Molecular Biology, TPC6
The Scripps Research Institute,
10550 North Torrey Pines Road,
La Jolla, CA 92037

E-mail: red...@scripps.edu
WWW:http://www.scripps.edu/~reddyv
VIPERdb:http://viperdb.scripps.edu

Phone:(858) 784-8191
FAX:(858) 784-8896

[ccp4bb] 2 post-doctoral positions in virus crystallography

[Pavel Plevka]

Dear all,

two post-doctoral positions are available in Pavel Plevka's group at Ceitec in 
the Czech Republic:
http://www.ceitec.eu/programs/structural-biology/x-ray-crystallography-ii/

The projects involve x-ray crystallography and cryo-electron microscopy of 
human viruses.

If you are interested, details of the jobs and how to apply can be found here:
http://www.ceitec.eu/new-position-post-doctoral-research-associate-2-positions-2/

All the best,
Pavel

Re: [ccp4bb] Chiral volume outliers SO4

[Robbie Joosten]

Hi Ian,

 
> > @Ian: You'd be surprised how well Refmac can flatten sulfates if you
> > have a chiral volume outlier (see Figure 1d in Acta Cryst. D68: 484-496
> (2012)).
> But this is only because the 'negative' volume sign was erroneously used
in
> the chiral restraint instead of 'both' (or better still IMO no chiral
restraint at
> all), right?  If so I don't find it surprising at all that Refmac tried to
flip the
> sulphate and ended up flattening it.
>  Seems to be a good illustration of the GIGO (garbage in - garbage
> out) principle.  Just because the garbage input in this case is in the
official
> CCP4 distribution and not (as is of course more commonly the
> case) perpetrated by the user doesn't make it any less garbage.
The problem is that in the creation of chiral volume targets chemically
equivalent (groups of) atoms are not recognized as such. So any new or
recreated restraint files will have either 'positiv' or 'negativ' and the
problem starts all over again. That is why it is better to stay consistent
and choose one chirality (the same one as in the 'ideal' coordinates in the
PDB ligand descriptions). This will also make it easier compare ligands
after aligning them (this applies to ligands more complex than sulfate).
Obviously, users should not be forced to deal with these things. Programs
like Refmac and COOT should fix chiral volume inversions for the user,
because it is only relevant inside the computer. That is the idea of chiron,
just fix these 'problems' automatically by swapping equivalent atoms
whenever Refmac gives a chiral volume inversion warning.  It should make
life a bit easier.


> The point I was making is that in this and similar cases you don't need a
chiral
> restraint at all: surely 4 bond lengths and 6 bond angles define the
chiral
> volume pretty well already?  Or are there cases where without a chiral
> restraint the refinement still tries to flip the chirality (I would fine
that hard to
> believe).
I agree with you for sulfate, and also for phosphate ;). I don't know what
happens in other compounds at poor resolution, when bond and angle targets
(and their SDs) are not equivalent. I guess that some angle might 'give way'
before others. That is something that should be tested. I have a growing
list of chiral centers that have this problem if you are interested.

Cheers,
Robbie

[ccp4bb] PhD position Diabetes

[Messias, Ana, Dr.]

Doctoral thesis in structure-based drug design in diabetes at the Helmholtz 
Zentrum München, Munich (Germany)

Job Details
The need for new and more effective drugs for type 2 diabetes is a major health 
concern. A doctoral thesis project is available at the Institute of Structural 
Biology. The main objective of this project is to discover new selective 
compounds targeting proteins relevant in type 2 diabetes using structure-based 
approaches.

Qualifications
-   We are looking for a highly motivated candidate who has interest in 
protein expression, structural biology (NMR and X-ray crystallography), 
diabetes and drug design.
-   Applicants should have a Diploma or Master’s degree in Biochemistry 
with an above-average grade, and practical experience in basic methods of 
molecular biology and protein biochemistry.
-   Previous experience in NMR, crystallography or drug design is a plus.
-   The candidate must be innovative, highly motivated, and with a strong 
interest in team work.
-   Strong communication and organizational skills are expected.

Inquiries should be addressed  to Dr. Ana Messias 
(ana.mess...@helmholtz-muenchen.de) or Prof. Michael Sattler 
(satt...@helmholtz-muenchen.de)


---
Dr. Ana Messias
Helmholtz Zentrum München
Institute of Structural Biology
(Building 43b, room 103)
Ingolstädter Landstr. 1
85764 Neuherberg
Germany

Tel: +49 (0)89 3187- 4706 (office) / - 3732 (lab)
E-mail: ana.mess...@helmholtz-muenchen.de
---

Helmholtz Zentrum München
Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH)
Ingolstädter Landstr. 1
85764 Neuherberg
www.helmholtz-muenchen.de
Aufsichtsratsvorsitzende: MinDir´in Bärbel Brumme-Bothe
Geschäftsführer: Prof. Dr. Günther Wess und Dr. Nikolaus Blum
Registergericht: Amtsgericht München HRB 6466
USt-IdNr: DE 129521671

Re: [ccp4bb] Chiral volume outliers SO4

[Ian Tickle]

On 11 July 2012 11:39, Robbie Joosten  wrote:

> @Ian: You’d be surprised how well Refmac can flatten sulfates if you have a
> chiral volume outlier (see Figure 1d in Acta Cryst. D68: 484-496 (2012)).

But this is only because the 'negative' volume sign was erroneously
used in the chiral restraint instead of 'both' (or better still IMO no
chiral restraint at all), right?  If so I don't find it surprising at
all that Refmac tried to flip the sulphate and ended up flattening it.
 Seems to be a good illustration of the GIGO (garbage in - garbage
out) principle.  Just because the garbage input in this case is in the
official CCP4 distribution and not (as is of course more commonly the
case) perpetrated by the user doesn't make it any less garbage.

The point I was making is that in this and similar cases you don't
need a chiral restraint at all: surely 4 bond lengths and 6 bond
angles define the chiral volume pretty well already?  Or are there
cases where without a chiral restraint the refinement still tries to
flip the chirality (I would fine that hard to believe).

Seems to be a case of "belt and braces"!

Cheers

-- Ian

Re: [ccp4bb] Chiral volume outliers SO4

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

In ccp4-6.3.0, the chiral volume of SO4 is marked as 'both'.
 SO4  chir_01  S  O1 O2 O3both

Tim

On 07/11/12 12:39, Robbie Joosten wrote:
> Hi Joel,
> 
> 
> 
> I prefer the swapping of atom names, which is pretty much what the
> program chiron does, over hacking the restraint file. The latter
> makes the problem reappear as soon as you use your PDB file on a
> machine with an 'unhacked' restraint file.
> 
> 
> 
> @Ian: You'd be surprised how well Refmac can flatten sulfates if
> you have a chiral volume outlier (see Figure 1d in Acta Cryst. D68:
> 484-496 (2012)).
> 
> 
> 
> Cheers,
> 
> Robbie
> 
> 
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
> Of Eleanor Dodson Sent: Wednesday, July 11, 2012 12:15 To:
> CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Chiral volume outliers
> SO4
> 
> 
> 
> Well - the problem may well be here - in the REFMAC dictionary
> (see $CLIBD/monomers.s.SO4.cif ) the chiral volume calculation uses
> at the order of the O numbering around the S atom . So if your O
> numbering is not right handed you will have the chiral volume 
> calculated as positive, not negative.
> 
> There are various fixes. Edit the SO4 and just the numbering of 2
> of the O atoms - eg use labels: S O2 O1 O3 Or edit the SO4.cif to
> have SO4  chir_01  S  O1 O2 O3 both
> 
> Eleanor
> 
> 
> loop_ _chem_comp_chir.comp_id _chem_comp_chir.id 
> _chem_comp_chir.atom_id_centre _chem_comp_chir.atom_id_1 
> _chem_comp_chir.atom_id_2 _chem_comp_chir.atom_id_3 
> _chem_comp_chir.volume_sign SO4  chir_01  S  O1 O2
> O3negativ
> 
> On 11 July 2012 10:59, Tim Gruene  wrote:
> 
> Dear Joel,
> 
> out of curiosity: what is "the Chiral volume outliers issue"?
> 
> Cheers, Tim
> 
> 
> On 07/11/12 01:00, Joel Tyndall wrote:
>> Hi people,
> 
>> We are refining a structure with sulfates and we are getting the 
>> Chiral volume outliers issue. I understand the problem as being 
>> computational where the oxygens are in reality equivalent but 
>> computationally named differently. I have seen the recent Acta 
>> Cryst D paper (April 2012 - PDB_REDO) which talks about this
>> issue and mentions the development of Chiron which could fix this
>> issue.
> 
>> Is there a way to fix this problem using existing tools (or 
>> editing).
> 
>> We have ~20 sulfates in our protein (10-mer system)
> 
>> Thanks heaps
> 
>> Joel
> 
>> _ Joel Tyndall, PhD
> 
>> Senior Lecturer in Medicinal Chemistry National School of
>> Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand
>> Skype: jtyndall
> 
>> Ph: +64 3 479 7293 
> 
> 
> 
> 
> 
> 
> 

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFP/Vt+UxlJ7aRr7hoRAu3aAJ9lBBav5Fz9W310QZCA+8hBrvYyEACfe/wV
a56yOVyYtgOODEN2ypSLMA0=
=9yvs
-END PGP SIGNATURE-

Re: [ccp4bb] sulphur bridge

[Allan Pang]

Dear Amr,

Same here. I didn't put DTT or any reducing agent in my buffer. I got  
crystals of this protein.


Allan

Quoting herman.schreu...@sanofi.com:


Dear Amr,

Every protein is different, so one cannot make a general remark.
However, in my experience disulfide bonds are normally not affected by
mercaptoethanol or DTT. I have had one protein where the disulfide bonds
were not made, likely because the protein was produced in E.coli.
However, the protein still crystallized.

Good luck!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of amro selem
Sent: Wednesday, July 11, 2012 10:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] sulphur bridge


Hallo every one,

i am working on one enzyme who has sulphur bridge , is using 2
mercaptoethnol or DTT  during handling this protein for crystallography
is not desirable .
best regards

Amr








--
Allan Pang

PhD Student

G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS

Phone number: 02078828480

Twitter: @xerophytes

Re: [ccp4bb] Chiral volume outliers SO4

[Robbie Joosten]

Hi Joel,

 

I prefer the swapping of atom names, which is pretty much what the program
chiron does, over hacking the restraint file. The latter makes the problem
reappear as soon as you use your PDB file on a machine with an 'unhacked'
restraint file.

 

@Ian: You'd be surprised how well Refmac can flatten sulfates if you have a
chiral volume outlier (see Figure 1d in Acta Cryst. D68: 484-496 (2012)).

 

Cheers,

Robbie

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Eleanor Dodson
Sent: Wednesday, July 11, 2012 12:15
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Chiral volume outliers SO4

 

Well - the problem may well be here - in the REFMAC dictionary  (see
$CLIBD/monomers.s.SO4.cif ) the chiral volume calculation uses at the order
of the O numbering around the S atom .
So if your O numbering is not right handed you will have the chiral volume
calculated as positive, not negative.

There are various fixes.
Edit the SO4 and just the numbering of 2 of the O atoms - eg use labels: S
O2 O1 O3 
Or edit the SO4.cif to have SO4  chir_01  S  O1 O2 O3
both

Eleanor


loop_
_chem_comp_chir.comp_id
_chem_comp_chir.id
_chem_comp_chir.atom_id_centre
_chem_comp_chir.atom_id_1
_chem_comp_chir.atom_id_2
_chem_comp_chir.atom_id_3
_chem_comp_chir.volume_sign
 SO4  chir_01  S  O1 O2 O3negativ

On 11 July 2012 10:59, Tim Gruene  wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Joel,

out of curiosity: what is "the Chiral volume outliers issue"?

Cheers,
Tim


On 07/11/12 01:00, Joel Tyndall wrote:
> Hi people,
>
> We are refining a structure with sulfates and we are getting the
> Chiral volume outliers issue. I understand the problem as being
> computational where the oxygens are in reality equivalent but
> computationally named differently. I have seen the recent Acta
> Cryst D paper (April 2012 - PDB_REDO) which talks about this issue
> and mentions the development of Chiron which could fix this issue.
>
> Is there a way to fix this problem using existing tools (or
> editing).
>
> We have ~20 sulfates in our protein (10-mer system)
>
> Thanks heaps
>
> Joel
>
> _ Joel Tyndall, PhD
>
> Senior Lecturer in Medicinal Chemistry National School of Pharmacy
> University of Otago PO Box 56 Dunedin 9054 New Zealand Skype:
> jtyndall
>
> Ph: +64 3 479 7293  
>
>

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFP/U5+UxlJ7aRr7hoRAqaUAKD4Eo2tqarIwbK6+mHIfYYHcyhAKQCgy02F
XK8ZYLNW3gI873nrtkZSv9E=
=ishS
-END PGP SIGNATURE-

 

[ccp4bb] Too many symmetry molecules LINKed in refmac 5.5.0109

[Martin Moche]

Dear colleagues,

When using the LINK command in refmac5 version 5.5.0109 between symmetry 
related molecules as below

LINK PDC B 1191.61O3'   DA B 125   1555   2555  p
LINKO3'   DA B 1251.61 PDC B 119   1555   3555  p
LINK PDG C 2091.61O3'   DT D 108   1555   3555  p
LINKO3'   DT D 1081.61 PDG C 209   1555   2555  p

the output looks incorrect.

For each link defined I also got the same link to the wrong symmetry molecule 
i.e. in addition the links above (apparently) I am also getting:

LINK PDC B 1191.61O3'   DA B 125   1555   3555  p
LINKO3'   DA B 1251.61 PDC B 119   1555   2555  p
LINK PDG C 2091.61O3'   DT D 108   1555   2555  p
LINKO3'   DT D 1081.61 PDG C 209   1555   3555  p

and these ones screws up the refinement.

Am I writing the symmetry operators in the wrong position? so that refmac5 
interprets the second operator as BOTH 2555 and 3555??

I am trying to repeat refinement of 3GBI using the same asymmetric unit and 
LINKs as the original authors did.

Without the links the refinement is ok however correctly inserted links would 
make it even better since I get bumps instead of a covalent bond at the LINK 
positions.

Best regards,
Martin





WARNING : chain BB   is cyclic ?

I am repeating the refinement of 3GBI


Martin Moche, Ph.D.
Head of Protein Crystallography
Karolinska Institutet
MBB/PSF
Scheeles väg 2
171 77 Stockholm
Sweden
phone: +46-8-524 868 43
mobile: +46-73-322 93 27
fax:+46-8-524 868 68
email:martin.mo...@ki.se

Re: [ccp4bb] sulphur bridge

[Herman . Schreuder]

Dear Amr,
 
Every protein is different, so one cannot make a general remark.
However, in my experience disulfide bonds are normally not affected by
mercaptoethanol or DTT. I have had one protein where the disulfide bonds
were not made, likely because the protein was produced in E.coli.
However, the protein still crystallized.
 
Good luck!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of amro selem
Sent: Wednesday, July 11, 2012 10:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] sulphur bridge


Hallo every one, 

i am working on one enzyme who has sulphur bridge , is using 2
mercaptoethnol or DTT  during handling this protein for crystallography
is not desirable .
best regards 

Amr

[ccp4bb] FE-Sulfur proteins

[Jan Rashid Umar]

Dear All,

I will be grateful to your suggestions about Iron-Sulfur cluster protein
purification. My colleague has some problems with the purification and it
seems that the iron-sulfur cluster might be deformed, and protein is
aggregated. The purification is done under aerobic (normal condition), and
is there some method to reintegrate iron sulfur cluster back into the
protein molecule under these conditions. Can anybody suggest some
literature, protocol or something that can improve the protein aggregation?

I look forward to hearing from you.

Best wishes,

Jan

Re: [ccp4bb] Chiral volume outliers SO4

[Eleanor Dodson]

Well - the problem may well be here - in the REFMAC dictionary  (see
$CLIBD/monomers.s.SO4.cif ) the chiral volume calculation uses at the order
of the O numbering around the S atom .
So if your O numbering is not right handed you will have the chiral volume
calculated as positive, not negative.

There are various fixes.
Edit the SO4 and just the numbering of 2 of the O atoms - eg use labels: S
O2 O1 O3
Or edit the SO4.cif to have SO4  chir_01  S  O1 O2
O3both

Eleanor


loop_
_chem_comp_chir.comp_id
_chem_comp_chir.id
_chem_comp_chir.atom_id_centre
_chem_comp_chir.atom_id_1
_chem_comp_chir.atom_id_2
_chem_comp_chir.atom_id_3
_chem_comp_chir.volume_sign
 SO4  chir_01  S  O1 O2 O3negativ

On 11 July 2012 10:59, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear Joel,
>
> out of curiosity: what is "the Chiral volume outliers issue"?
>
> Cheers,
> Tim
>
> On 07/11/12 01:00, Joel Tyndall wrote:
> > Hi people,
> >
> > We are refining a structure with sulfates and we are getting the
> > Chiral volume outliers issue. I understand the problem as being
> > computational where the oxygens are in reality equivalent but
> > computationally named differently. I have seen the recent Acta
> > Cryst D paper (April 2012 - PDB_REDO) which talks about this issue
> > and mentions the development of Chiron which could fix this issue.
> >
> > Is there a way to fix this problem using existing tools (or
> > editing).
> >
> > We have ~20 sulfates in our protein (10-mer system)
> >
> > Thanks heaps
> >
> > Joel
> >
> > _ Joel Tyndall, PhD
> >
> > Senior Lecturer in Medicinal Chemistry National School of Pharmacy
> > University of Otago PO Box 56 Dunedin 9054 New Zealand Skype:
> > jtyndall
> >
> > Ph: +64 3 479 7293
> >
> >
>
> - --
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
>
> iD8DBQFP/U5+UxlJ7aRr7hoRAqaUAKD4Eo2tqarIwbK6+mHIfYYHcyhAKQCgy02F
> XK8ZYLNW3gI873nrtkZSv9E=
> =ishS
> -END PGP SIGNATURE-
>

Re: [ccp4bb] Chiral volume outliers SO4

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Joel,

out of curiosity: what is "the Chiral volume outliers issue"?

Cheers,
Tim

On 07/11/12 01:00, Joel Tyndall wrote:
> Hi people,
> 
> We are refining a structure with sulfates and we are getting the
> Chiral volume outliers issue. I understand the problem as being
> computational where the oxygens are in reality equivalent but
> computationally named differently. I have seen the recent Acta
> Cryst D paper (April 2012 - PDB_REDO) which talks about this issue
> and mentions the development of Chiron which could fix this issue.
> 
> Is there a way to fix this problem using existing tools (or
> editing).
> 
> We have ~20 sulfates in our protein (10-mer system)
> 
> Thanks heaps
> 
> Joel
> 
> _ Joel Tyndall, PhD
> 
> Senior Lecturer in Medicinal Chemistry National School of Pharmacy 
> University of Otago PO Box 56 Dunedin 9054 New Zealand Skype:
> jtyndall
> 
> Ph: +64 3 479 7293
> 
> 

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFP/U5+UxlJ7aRr7hoRAqaUAKD4Eo2tqarIwbK6+mHIfYYHcyhAKQCgy02F
XK8ZYLNW3gI873nrtkZSv9E=
=ishS
-END PGP SIGNATURE-

Re: [ccp4bb] PS: Chiral volume outliers SO4

[Ian Tickle]

HI Joe

This and similar cases should be changed in the library from
_chem_comp_chir.volume_sign = 'negative' (or 'positive') to 'both'.
Then if you're concerned about the standardisation of the atom
labelling you can always swap the labels of 2 atoms in the PDB file,
but at least with 'both' non-standard labelling won't mess up the
refinement.  In this particular case the chiral specification seems
somewhat redundant anyway since the geometry is completely nailed down
by the 6 angle restraints! - so an even better solution might be to
delete the _chem_comp_chir block entirely.  You only need chiral
restraints if one atom is a H (unless I suppose the density for the
4th atom is very poor).  It would seem unlikely that the angle
restraints would allow 'umbrella flips' if all 4 are non-H.

Cheers

-- Ian

On 11 July 2012 00:02, Joel Tyndall  wrote:
> Sorry I should have added, that the issue occurs following refinement in
> refmac. (error in log file and SO4’s are distorted)
>
>
>
> _
>
> Joel Tyndall, PhD
>
> Senior Lecturer in Medicinal Chemistry
> National School of Pharmacy
> University of Otago
> PO Box 56 Dunedin 9054
> New Zealand
>
> Skype: jtyndall
>
>
>
> Ph: +64 3 479 7293
>
>

Re: [ccp4bb] Does anyone have used DelPhi to calculate the Electrostatic potentials of DNA ？

[Steiner, Roberto]

Dear Eleanor

I (capital!) believe the original poster means 
http://compbio.clemson.edu/delphi.php

Best wishes
Roberto

On 11 Jul 2012, at 09:54, Eleanor Dodson wrote:


On 11 Jul 2012, at 03:36, dengzq1987
 don't understand the question really.  What DELPHI do you mean?

Many programs output DELPHI (refmac,  SigmaA etc ) and they are usually used as 
input to a difference map using FFT
Eleanor


Hi all,
recently，I want to use DelPhi to calculate the Electrostatic potentials of 
DNA.but in the manual,i can not find the method to create the inputfile fort.11 
、fort.12 and fort.13.Does anyone have experience on this?  Please suggest

Thank you in advance

Sincerely
dengzq


Roberto Steiner, PhD
Group Leader
Randall Division of Cell and Molecular Biophysics
King's College London

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL, London, UK
Tel 0044-20-78488216
Fax 0044-20-78486435
roberto.stei...@kcl.ac.uk

[ccp4bb] Punctuation etc.

[Patrick Shaw Stewart]

Could I make a request?

Could people who ask questions to the bb possibly lay out their questions
clearly, capitalize the word "I" and use the correct punctuation such as
question marks?

It makes it so much easier to read, and - this is more important -  you
will get more helpful responses if you help people to understand what
you're asking.

Giving your message a meaningful title is also very helpful!

Best wishes to all,

Patrick


-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] Does anyone have used DelPhi to calculate the Electrostatic potentials of DNA ？

[Eleanor Dodson]

On 11 Jul 2012, at 03:36, dengzq1987 
 don't understand the question really.  What DELPHI do you mean?

Many programs output DELPHI (refmac,  SigmaA etc ) and they are usually used as 
input to a difference map using FFT
Eleanor


> Hi all,
> recently，I want to use DelPhi to calculate the Electrostatic potentials of 
> DNA.but in the manual,i can not find the method to create the inputfile 
> fort.11 、fort.12 and fort.13.Does anyone have experience on this?  Please 
> suggest
> 
> Thank you in advance
> 
> Sincerely
> dengzq

[ccp4bb] sulphur bridge

[amro selem]

Hallo every one, 

i am working on one enzyme who has sulphur bridge , is using 2 mercaptoethnol 
or DTT  during handling this protein for crystallography is not desirable .
best regards 

Amr

[ccp4bb] Workshop on Advanced Protein Engineering Techniques & 3rd P-CUBE User Meeting, Heidelberg, 22-24 Oct 2012

[Darren Hart]

Workshop on Advanced Protein Engineering Techniques & 3rd P-CUBE User
Meeting  Heidelberg, 22 - 24 October 2012*
*
* Please apply here:  www.p-cube.eu*

Registration deadline: 10 August 2012

*3rd P-CUBE User Meeting (22 Oct 2012)*

Get to know everything about the numerous platforms of P-CUBE and listen to
the platform leaders presenting their infrastructure (Protein Expression in
Bacterial & Eukaryotic Cells, MultiBac, ESPRIT, DARPins, High-throughput
Crystallization, Advanced Microscopy). If you are a former user of one of
the platforms, you might even get selected to speak about your data.  All
the others will have the opportunity to present posters. Please register
online for the P-CUBE User Meeting at www.p-cube.eu.

*Workshop on Advanced Protein Engineering Techniques (23 - 24 Oct 2012)*

The Workshop on Advanced Protein Engineering Techniques is targeted at
early career scientists (PhD student/Postdoc), who are interested in
learning the basics of advanced protein engineering techniques.

 The hands-on course will be taught in small groups and will cover the
following topics:

•Genetically encoding unnatural amino acids via amber
suppression for site-specific protein engineering

•Advanced protein engineering with intein technology

Speakers include:

Stefan Knapp, Oxford University, UK (Keynote Lecture)

Luc Brunsveld, TU Eindhoven, NL
Maja Köhn, EMBL Heidelberg, DE

Edward Lemke, EMBL Heidelberg, DE

Henning Mootz, Muenster University, DE

Daniel Summerer, Konstanz University, DE


To apply for the P-CUBE User Meeting and the workshop please visit our web
page at www.p-cube.eu. Scientists applying for the workshop will be asked
to submit their CV as well as a letter of motivation. A maximum of 12
participants will be selected for the workshop. The meeting & workshop will
be open to scientists from EU Member and/or Associated States.

*General Information*

•There is no registration fee.

•Lunch & 2 dinners will be paid by P-CUBE.

•Accommodation will be covered for workshop participants as
well as selected speakers at the user meeting in the Hotel ISG (shared
rooms, 3 nights max. workshop participants) and the Exzellenz Hotel (shared
rooms, 2 nights max. user meeting speakers).

•Participants are responsible for their own travel costs.
However, travel costs of speakers at the user meeting will be partially
reimbursed (300 € max.). For workshop participants there is the possibility
to apply for a small number of travel grants (300 € max.).

*Venue*

The 3rd P-CUBE User Meeting and the workshop will take place at EMBL
Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany. Please visit this
webpage: www.embl.de

*For more information, please contact Petra Lindemann (
petra.lindem...@embl.de) or Jutta Tatzel (j.tat...@bioc.uzh.ch)*

We look forward to welcoming you in Heidelberg!
Markus Grütter (University of Zurich, CH)
Maja Köhn (EMBL Heidelberg, DE)
Edward Lemke (EMBL Heidelberg, DE)




Dr. Petra Lindemann
Scientific Administrator

Mueller (Christoph) Group
EMBL Heidelberg
Meyerhofstraße 1
69117 Heidelberg
Germany
phone: +49 6221 387- 8365