X-ray equipment available

From: Steiner, Roberto
Date: 31 March 2012 15:45

Full Rigaku R-axis IIC  Image Plate X-ray diffraction system

We would like to offer a complete macromolecular diffraction system free of charge to anyone prepared to pay (up-front) the shipping costs from our laboratory to theirs.

The system consists of a R-axis IIC area detector, Rigaku RU-200BH rotating anode X-ray generator with Yale mirrors together with an Oxford cryosystems 600 series cryostream and associated dewars.  The system includes a purpose built enclose made of lead-rated perspex, obviating the need for a walk in enclosure.  The footprint of the X-ray generator cabinet 810cm (D) x 1100cm (W) x 900cm (H),  the footprint of the radiation enclosure is 1400cm (D) x 1750cm (W) x 1000cm (H). The weight of the X-ray generator is  (550kg) and the HV transformer (450kg), RaxisIIC (150kg), enclosure (80kg) and table (80kg).

We wish to give away the whole system and not break it down to parts, so requests should be for the complete system.

Please contact Marty Rajaratnam (Randall division of Cell and Molecular Biophysics, King's College London) email r.rajaratnamATkcl.ac.uk if you are interested in acquiring this system.

With best wishes

_ 

Roberto Steiner, 

a small trick for protein and organic compound cocrystallization.

From: Kevin Jin
Date: 30 March 2012 09:02
Dear All,

Here is way I have used for protein and hydrophobic organic compound
cocrystallization. Via this method, less amount of DMSO will be used
to aviod protein ppt.

I hope you can use for your research.

http://www.jinkai.org/Xtal.html

Regards,

--
Kevin Jin

Sharing knowledge each other is always very joyful......

Website: http://www.jinkai.org/

----------
From: Bryan Lepore
On Fri, Mar 30, 2012 at 4:02 AM, Kevin Jin <kevinjin@gmail.com> wrote:
> Here is way I have used for [...]

I hate to be a curmudgeon, but can a list member please explain why
this is not specifically blogspam or spam - or whatever it is exactly?


-Bryan

----------
From: Jacob Keller
What's the harm? Seems relevant to crystallographers, and not for self-promotion, but just to help, share an interesting tip. Perhaps you can think of it as a response to an un-asked but plausible question, i.e., "how can I treat my coverslips to make them more receptive to organic solvent-containing drops?

Jacob

ps I bet you don't really hate to be a curmudgeon--there is a certain pleasure in it!

--
*******************************************
Jacob Pearson Keller

----------
From: Bryan Lepore
On Fri, Mar 30, 2012 at 3:26 PM, Jacob Keller
 wrote:
> What's the harm?

have you seen usenet/Google groups?

-Bryan

----------
From: James Stroud

It's not really "spam", but there are better channels to communicate these type of updates. Twitter is probably the best known channel. People get to subscribe and authors can push their contributions to willful and interested subscribers. The author posts a summary of what's behind the link and then posts the link, just as Jin is doing with his ad-hoc feed here.

For example, a few days ago I created the @amyloids twitter feed (https://twitter.com/#!/amyloids) that features recent publications I have read and that I also think are of interest to amyloid researchers.

Instead of being to critical, let's just direct authors to use the proper channels. Social media has moved fast in recent years, and we should be understanding as the scientific community plays catch-up.

If Jin created a twitter feed for tips, it would probably get some subscribers from this board--as long as we don't begrudge the Jin an opportunity to send out an advertisement for the feed itself.

James

----------
From: Zhijie Li
Hi,

If those are spams, then Kevin must be the most successful spammer I have ever encountered, as I have actually read all his posts and linked web pages.

About the twitter idea, I am not sure if I would ever become a twitter user in the near future. Compared to following tens of authors, I think a quick glance at the CCP4BB would remain a much more efficient means for learning about new things. There is also a filtering function in my email client program - certainly beats twitter/FB/groups IMHO.

Zhijie

processing data with ice ring

From: Shanti Pal Gangwar
Date: 30 March 2012 05:30

  Hello all,

I have a query regrading processing of data.  I have  good diffraction data that  has ice rings. Is it possible to mask the regions of the ice rings during processing?
I have been using Mosflm to process data.  
Is this feature available with this program? 

Suggestions and advice would be greatly appreciated.

Thanks


regards

SHANTI PAL






----------
From: Sita Ram Meena
Hi Shanti

Mosflm has a default option in the "processing options" menu.

when you runs imosflm  go to the setting and than processing option.

setting => processing options.
This has five tabs 1. Spot finding, 2. indexing, 3. Processing 4. Advance Processing, 5. Advance integration.

initial three (1, 2, 3) tab has has ice ring exclusion options , you just need to check them that's it.

1. In the Spot finding => Automatic ice and Powder ring exclusion.
2. In Indexing => Exclude any spot rings.
3. Processing => Do not process spots near ice rings.

This will exclude your ice ring but you may loose some information from your data.

Good luck

--
Sita Ram

----------
From: Harry Powell

Hi

Actually, it's *much* easier than this in iMosflm (though Sita's suggestion is already pretty easy). There's no need to open the "Processing Options" menu in this case.

On each of the indexing,  the refinement and integration panes, there is a small button on the top row of widgets with what looks like a snow flake - click on this to activate ice ring exclusion for processing the data.

As Sita says, you will lose some information about genuine non-ice spots on (or very near to) the ice rings, but this is generally more than compensated for by the improved processing of the spots you actually do integrate.

Harry

--

Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH

----------
From: Loes Kroon-Batenburg

Dear Shanti,

You may want to try EVAL (www.crystal.chem.uu.nl/distr/eval). It provides options to exclude regions affected with ice diffraction, or practically any region you'd like. In our experience data treated in this way have a lower Rmerge. However, the difference with respect to refinement statistics is not all that large, because ice-affected reflections are mostly rejected in the scaling step if you have sufficient redundancy. Ice scattering is rarely constant over theta-rings.

Regards,
Loes.

--
_
__________________________________________

about heavy atom derivatization

From: Shanti Pal Gangwar
Date: 30 March 2012 14:34
Dear all

I am beginner in crystallography.We have collected a native data of a given protein at 2.2A resolution but are unable t solve by MR
 therefore we collected Hg, and Pt derivative after  soaking the crystals. I don't know how to process this heavy atom derivative data
and find the anomalous signal if heavy atom is there?

thanks in advance for help and suggestions..


regards

Shanti Pal Gangwar



----------
From: Ed Pozharski
http://www.ccp4.ac.uk/dist/examples/tutorial/html/heavy-tutorial-mir.html

seems relevant

--
Edwin Pozharski, PhD, 

----------
From: Francis E Reyes
To add to Ed's comments:


Find someone in the immediate department/area to walk you through your first structure.


But to answer your question:

What about the MR didn't work?

Use your MR model to find your heavy atoms. They can be found using anomalous data  difference fouriers (or even better log likelihood gradient maps) using data  as low as 6-8A, and, in my case, using a model that was about 50% (mass) of the ASU with the placement of that 50% to be about 80-90% correct.


F

---------------------------------------------
Francis E. Reyes M.Sc.

Lectureship in Biochemistry, University of Bath - UK

Date: 30 March 2012 05:43


Lectureship in Biochemistry
(Physical Biochemistry of Molecular Interactions)
Department of Biology and Biochemistry
University of Bath, Claverton Down
Bath BA2 7AY, UK

We seek an outstanding scientist with an established track record of independent research in biochemistry, and in particular in the physical biochemistry of molecular interactions. The person appointed will be expected to establish and sustain a rigorous externally funded research programme and to have a commitment to excellent teaching. Applications are particularly welcome from candidates who have the potential to develop and sustain collaborations with academics in our Department research themes and to foster collaboration across the University and beyond.

The Department of Biology & Biochemistry is one of the leading research intensive Biosciences departments in the UK. The outstanding quality of our research is reflected in the level of external funding from the UK Research Councils, medical charities, European Union and Industry as well as publications in the top international journals (Nature, Science, PNAS and PLoS Biology).

Informal enquiries may be made to the Head of Department, Dr. Richard Hooley  but please ensure that applications are submitted through the University of Bath Website. Further details about the position are available at-

http://www.bath.ac.uk/jobs/Vacancy.aspx?ref=JK983

Application closing date: 30 APRIL 2012

postdoctoral position in structural neurobiology

Date: 28 March 2012 22:12

A postdoctoral position at Yale University School of Medicine is available immediately for highly motivated, creative individuals with a strong interest in the structure, function, and pharmacology of signaling proteins implicated in neurological and neuropsychiatric diseases. We are presently focusing our efforts on central nervous system transporters using a combination of steady-state kinetics, radioligand binding, X-ray crystallography, and other complementary biochemical/biophysical techniques.

 

The laboratory is located in newly-renovated space in the Department of Cellular and Molecular Physiology in a highly collaborative environment. We have state of the art tissue culture facilities, purification equipment, and LINUX workstations. We have regular access to Mosquito and Art Robbins Gryphon LCP (lipidic cubic phase) crystallization robots, Formulatrix imager with UV detection, in-house X-ray diffractometer, synchrotron beamlines, and number of biophysical instruments. These include a CD spectrometer, analytical ultracentrifuge, differential scanning calorimeter, isothermal titration calorimeter, surface plasmon resonance, static/dynamic light scattering, and stopped flow fluorometer.

 

Candidates must hold (or soon expect to hold) a Ph.D. in biochemistry, biophysics, or a related field and have a strong publication record. Experience in recombinant DNA methods, protein expression and purification, and X-ray crystallography is required. Prior work with membrane proteins, insect/mammalian expression systems, and functional assays is preferred. The candidate must have an excellent command of oral and written English. He/she must be capable of working independently but also be willing to work as part of a team.

 

For more information, please contact me via e-mail.

 

Applicants should send a CV, along with a summary of previous research experience and interests, and arrange to have 3 reference letters sent to Satinder K. Singh via e-mail at satinder.k.singh at yale.edu

 

Additional contact information is given below:

 

Satinder K. Singh, Ph.D.

Website: http://physiology.yale.edu/people/satinder_k_singh-3.profile

 

Postdoc/PhD positions available in Bristol

Date: 28 March 2012 10:13

Dear All,

      please see below for links detailing two positions currently available here in Bristol, 1x 2 yr PDRA position in natural product enzymology, and 1x 3 yr PhD studentship in the structural biology of strep virulence. The former would ideally suit a postdoctoral level scientist with a strong track record in molecular biology and protein expression/purification, ideally allied to either PX, NMR, or EM. The latter would suit an enthusiastic graduate (or soon to be) with a 2.i or better in biochemistry, microbiology, or a related discipline, with an interest in applying structural methods to the study of bacterial pathogenicity.  Both projects will involve significant collaborative work with other Bristol based researchers and as such the ability to effectively communicate with individuals from varying scientific disciplines is essential.

 Informal enquires welcome, though full applications MUST be made via the UoB links provided.

 Cheers,

           Paul

Studentship

http://www.findaphd.com/search/ProjectDetails.aspx?PJID=38069&LID=190

Postdoc

http://www.bris.ac.uk/boris/jobs/feeds/ads?ID=109391

http://www.bris.ac.uk/biochemistry/research/pr.html

Postdoctoral position in structural biology at the ISMB, London

Date: 29 March 2012 12:13

A postdoctoral position is currently available to work in the laboratory of Dr. Cara Vaughan at the Institute of Structural and Molecular Biology, Birkbeck College, London, UK. The aim of the project is to investigate the structure of Hsp90 cochaperone and client complexes using both protein crystallography and electron microscopy. The position is for 3 years and is funded by the BBSRC. Further information on the group's research interests and relevant publications can be found at http://people.cryst.bbk.ac.uk/~ubcg72a/Home.html

 

Applicants must have a PhD in Structural Molecular Biology or a relevant related field. Experience of multi-protein complex expression and purification, protein crystallography, single particle electron microscopy, or experience of yeast genetics and relevant research publications will be an advantage.

 

To apply for this position please go to www.bbk.ac.uk/jobs and search using reference number 10711.
Closing data for completed applications: 8 May 2012

42nd Mid-Atlantic Macromolecular Crystallography Meeting

From: David R. Cooper
Date: 29 March 2012 22:14

42^nd Mid-Atlantic Macromolecular Crystallography Meeting

University of Virginia, Charlottesville, VA

May 31 – June 2, 2012

The 42^nd Mid-Atlantic Macromolecular Crystallography Meeting (MAMCM) will be held at the University of Virginia in Charlottesville, Virginia, from May 31^st through June 2^nd, 2012.  The MAMCM serves as a platform for the presentation of the latest research results and technical advances, and as a forum to facilitate discussions amongst structural biologists in the mid-Atlantic region. Registration is now open at http://www.mid-atlantic.org.  There is no registration fee.

This year the keynote speaker will be Dr. William Weis from the Stanford University School of Medicine. The meeting will also feature two interactive workshops: a Structure Determination Workshop led by Drs. Chruszcz and Cooper of Dr. Wladek Minor's laboratory and featuring Dr. Jim Pflugrath, and a Lipidic Cubic Phase (LCP) Crystallization Workshop led by Dr. Vadim Cherezov of The Scripps Research Institute.

Platform presentations will be selected from submitted abstracts. Preference will be given to young researchers (students and fellows), and a Student/Fellow Poster Prize(s) will also be awarded.

We have blocked off rooms at several local hotels, as well as conveniently located suites provided by University Housing.

IMPORTANT: The MAMCM coincides with a reunion weekend at the University of Virginia.  Please reserve your accommodations early!  Note that most of the blocked rooms will be released on April 30th.

http://www.mid-atlantic.org

Respectfully,

Drs. David Cooper, Peter Horanyi and Michael Purdy

Co-Directors of the MAMCM

Postdoctor​al position in Grenoble (France)

From: Antoine Royant
Date: 28 March 2012 15:32


POSTDOCTORAL POSITION - INSTITUT DE BIOLOGIE STRUCTURALE, GRENOBLE, FRANCE

POSITION: The Structural Protein Dynamics group at the Institut de Biologie Structurale in Grenoble is looking for a highly motivated researcher to join the team as a postdoc to work for 30 months on a project funded by the Young Scientist program of the French National Research Agency ANR. The gross salary will be 2500-2900 €/month, depending on experience.

RESEARCH: The aim of the project is primarily the characterization of protein damage induced by singlet oxygen (a reactive oxygen species) by combination of X-ray crystallography, optical spectroscopy and mass spectrometry. It also involves the structural characterization of genetically-encoded systems capable of either sensing or detecting singlet oxygen, which should provide information for further rational evolution of the systems. Some recent publications relevant to the project are Shu et al. Science (2009), Shu et al. PLoS Biol. (2011), Goedhart et al. Nat. Commun. (2012).

SCIENTIFIC ENVIRONMENT: The work will be performed at the Institut de Biologie Structurale (IBS) in Grenoble, at the foot of the French Alps. It will involve the use of the in crystallo spectroscopy lab Cryobench and of the X-ray beamlines of the European Synchrotron Radiation Facility (ESRF), and benefit from the strong background in structural biology provided by the PSB initiative which includes IBS, ESRF, ILL, EMBL-Grenoble and UVHCI.

APPLICATION: The successful candidate should have a PhD in biology with a strong background in molecular biology and biochemistry. Previous experience with protein X-ray crystallography or optical spectroscopy will be favourably considered. Applications and informal queries should be addressed to Antoine Royant (antoine.royant @ ibs.fr). Applicants should send a CV and a cover letter describing their research interest and including the names and e-mail addresses of persons willing to write a recommendation letter.

CONTACT: Antoine Royant
Structural Protein Dynamics group
Institut de Biologie Structurale
41 rue Jules Horowitz
38027 Grenoble cedex 1
France

dear all

From: rana ibd
Date: 28 March 2012 13:40

Dear all 

yes my paalet is large enough and no my protein is not soluble ,and  I am using 37degrees before and after induction with IPTG,and the vector that I am using is pRSETA 3.2kd

Best Regards

Rana

---

From: Kelly Daughtry
Sent: Tuesday, March 27, 2012 5:45 PM
Subject: Re: [ccp4bb] dea all

When you lyse the cells and spin down cellular debris, is the pellet large and white (indicating inclusion bodies)? Is your protein soluble or membrane? What temperature did you use for expression? What vector are you using? Providing more details allows us to better answer your questions.

Off the top of my head:

Altering expression can include lowered the temperature just prior to induction (25, 18, or lower) and letting the cells grow overnight. Induction at increased cell density (1.0 vs 0.6 O.D.).

Anther option to increase the expression of soluble protein is to use the auto-induction media: http://www.ncbi.nlm.nih.gov/pubmed/15915565 

Another option is to try other cells lines, and co-expression with chaperonins (the arctic express cell line is useful  http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=467  ).

Did you try other tags (GST, MBP, etc)?

Hope this helps,

Kelly Daughtry

*******************************************************
Kelly Daughtry, Ph.D.

On Tue, Mar 27, 2012 at 10:35 AM, rana ibd wrote:

Dear all

I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB madia, I am having problems with the expression which shows small amount of the protein , I also have problems with purification using NI-NTA by also having small amount even after extensive buffer exchange , Is it likely due to the small amount of protein in the medium , should I use a different kind of media, any sugestions or any kind of details or a paper that might help I will be thankful

Best Regards

Rana

Coot set-refine-max-residues

From: Morten Grøftehauge
Date: 27 March 2012 14:50
Hi ccp4bb,

I've been having trouble with Coot. Specifically the scripting to change the maximum number of residues to refine.
So if I try to refine more than 20 residues I get this neat little warning message in the terminal:

WARNING:: Hit heuristic fencepost! Too many residues to refine
           FYI: 23 > 20 (which is your current maximum).
Use (set-refine-max-residues 40) to increase limit

but when I try set-refine-max-residues 40 or set-refine-max-residues 30 in either the terminal or the scheme scripting command window, it doesn't actually change anything.
I've run the Python version of the hydrogen bond restraints script before all this.
Hope someone can point me in the right direction.

Cheers,
Morten

-- 
Morten K Grøftehauge, PhD 

Pohl Group

Durham University

----------
From: Ed Pozharski
works here on 0.7-pre-1 rev 3713

so try downloading the latest version if yours is <3713

On Tue, 2012-03-27 at 14:50 +0100, Morten Grøftehauge wrote:
> set-refine-max-residues
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
                                               Julian, King of Lemurs

Clustal omega

From: Jayashankar
Date: 27 March 2012 09:18

Dear All,

A bit offtopic question,

Does any body know how can I get the PIR output of the aligned sequence from clustal omega.
I have a full length protein solved, to give weightage during comparitive modelling, I use domains of the same/similar protein solved before.
I expect an alignment something that looks like one below, and clustalw does'nt seems to work , but clustal omega does it. But there is no .pir format output.


1.cccccccccccccaaaaaaaaaabbbbbbbb
2.-------------------------------------bbbbbbbb
3.ccccccccccccc------------------------------
4.------------------aaaaaaaaaaa--------------


Thanks
S.Jayashankar

----------
From: Tim Gruene

Dear S. Jayashankar,

have you tried the clustalw options '-convert -output=PIR'? This should
result in a pir-formatted output file.

Tim

- --
- --
Dr Tim Gruene

----------
From: Jayashankar

Dear Tim,

I try to model a full length protein based on various domains solved already.
In that case, alignment of multiple sequence should approximately locate to the corresponding domain region.

Clustalw results in an unexpected alignment file.
Whereas the output from clustal omega is perfect and the format what I need is not given in the output option.

Because I will be needing this .pir output file to model my target sequence using modeller.

thanks

S.Jayashankar

----------
From: Tim Gruene 

Dear Jayashankar,

sorry, you did explain this in your model and I flipped clustalo with
clustalw. Even though when I used the option '-convert', clustalw does
not re-align the input but simply converts it, so in case it can read
the clustalo output, this should do the job, shouldn't it?

Have you tried reading the output file with clustalx or seaview or
jalview? These programs should be able convert to various file formats
without messing up with the alignment.

Cheers,
Tim

----------
From: Jayashankar

Dear Tim,

Thanks, The Jalview, output to textbox in PIR worked thr trick.

S.Jayashankar

24 well screw-cap crystallization plates

From: Brad Bennett
Date: 27 March 2012 16:01
Hi all-

So far, I have not been a fan of Qiagen's decision to revamp the 24 well Nextal crystallization plates with screw caps to 15 well plates without a top cover plate. I know that it is less plastic and now a standard SBS footprint but IMHO it is rather flimsy and harder to handle than the original plates. Does anyone know of a distributor of the 24 well version of these plates or an alternative vendor that is manufacturing plates similar to these?

Thanking you in advance for any suggestions-

Brad

----------
From: Pascal Egea

Hi Brad,

I am afraid that there is no alternate source for these plates. The screw cap system , I believe, was patented by the canadian company NEXTAL that was then assimilated by Q...N and the patent is probably still holding. 

Pascal

--
Pascal F. Egea, PhD

For 24 well Netxal plates (non ccp4 question) ==>> SUMMARY

From: Mathews, Irimpan I.
Date: 27 March 2012 16:48


Hi Brad,

See below my attempts on this in 2010.

Regards,
Mathews

-----Original Message-----
From: Mathews, Irimpan I.
Sent: Friday, May 21, 2010 8:23 AM
Subject: RE: For 24 well Netxal plates (non ccp4 question) ==>> SUMMARY

Dear All,

Thank you for the several responses. Please see the summary below.

1. Several of them:  complain to Qiagen (two or three said: protests loudly).  Unfortunately, my e-mail to the Qiagen marketing manager never got any reply. I e-mailed to him on 5-12-10. Let me know if you need his e-mail address.

2.  Molecular dimensions has the following new item.

a novel 96 well version of a screw top plate for hanging drop which is of standard 96 well plate size.
Please see
http://www.moleculardimensions.com/shopdisplayproducts.asp?id=54&cat=Hanging
+drop+

Kind regards,
Mathews


-----Original Message-----
From: CCP4 bulletin board [ On Behalf Of Mathews, Irimpan I.
Sent: Tuesday, May 11, 2010 12:41 PM
Subject: [ccp4bb] For 24 well Netxal plates (non ccp4 question)

Dear All,

Qiagen has stopped selling the 24 well Netxal trays and changed to 15 well trays (almost same price) that doesn't fit with any screens. Do you know of some source for the 24 well screw cap trays?

Thank you very much for your help,
Mathews

dea all

From: rana ibd
Date: 27 March 2012 15:35

Dear all

I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB madia, I am having problems with the expression which shows small amount of the protein , I also have problems with purification using NI-NTA by also having small amount even after extensive buffer exchange , Is it likely due to the small amount of protein in the medium , should I use a different kind of media, any sugestions or any kind of details or a paper that might help I will be thankful

Best Regards

Rana

----------
From: Kelly Daughtry

When you lyse the cells and spin down cellular debris, is the pellet large and white (indicating inclusion bodies)? Is your protein soluble or membrane? What temperature did you use for expression? What vector are you using? Providing more details allows us to better answer your questions.

Off the top of my head:

Altering expression can include lowered the temperature just prior to induction (25, 18, or lower) and letting the cells grow overnight. Induction at increased cell density (1.0 vs 0.6 O.D.).

Anther option to increase the expression of soluble protein is to use the auto-induction media: http://www.ncbi.nlm.nih.gov/pubmed/15915565 

Another option is to try other cells lines, and co-expression with chaperonins (the arctic express cell line is useful  http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=467  ).

Did you try other tags (GST, MBP, etc)?

Hope this helps,

Kelly Daughtry

*******************************************************
Kelly Daughtry, Ph.D.

----------
From: Kevin Jin
I guess you already run SDS-PAGE to check the pellets before and after
sonication. Not only the media.

--
Kevin Jin

Candle always burns itself out to light up the tunnel .....

Website: http://www.jinkai.org/

Human multi-pass integral membrane protein crystal structures

From: Raji Edayathumangalam
Date: 3 March 2012 23:13


Dear fellow CCP4bb-ers,

Does anyone know how many crystal structures have been published to date for unique human multi-pass alpha-helical integral membrane proteins? For example, if there are multiple structures of the same membrane proteins with different ligands, I do not wish to count them as unique for my purposes.

I have already tried to search for this information on Stephen White's database, on Caffrey's page, in the PDB database, Google etc. but my searches either return no results or incomplete results. I am looking for a specific subset of membrane proteins that satisfy all following criteria: "human + multi-pass + alpha-helical + integral membrane protein."

If anyone can provide the answer, that would be very helpful. I need this information for a fellowship application.

Thanks much.

Raji

--
Raji Edayathumangalam

----------
From: Sean Seaver


Hi Raji,

Here is an approach that I hope helps:

Used the following pages:

http://opm.phar.umich.edu/superfamilies.php?class=1
http://opm.phar.umich.edu/superfamilies.php?class=16

Export/sort by species the second table using your favorite spreadsheet program:
-writing a script for this step maybe helpful

Generates the following list of 96 PDB entries for humans:
http://bit.ly/widcnV (link to Google docs page)

I took a look at each entry and added a short note which is worth double checking.

If you do not want to include structures solved by NMR or entries that differ by additional domains then editing will be needed.  Some entries listed as alpha helical do contain varying amounts of beta sheets which may or may not work for your purposes.

Maybe worth cross referencing with the following database:
http://pdbtm.enzim.hu/?m=sform
(search by alpha helical, transmembrane segments set to 2+ then find all results containing the term human)
note: this output includes theoretical models from PDB, which I suspect you may want to remove

Hopefully, someone will follow up with a more elegant solution and all the best with your application.

Take Care,

Sean Seaver

P212121
http://store.p212121.com/

COURSE ANNOUNCEMENT - BIOCRYS 2012

Date: 27 March 2012 11:49

Dear colleagues,

On behalf of the organizers, Maria Armenia Carrondo and Thomas R. Schneider I have the following course announcement:

*COURSE ANNOUNCEMENT - BIOCRYS 2012*
FEBS Practical & Lecture Course
Fundamentals of Modern Methods in Biocrystallography


20th - 27th October 2012 at the Instituto de Tecnologia Química e Biológica, Oeiras, Portugal.

Topics of the course will run from fundamentals such as symmetry, point groups and crystal systems, basic diffraction physics, reciprocal space and the Ewalds sphere, radiation damage, data processing, symmetry in the diffraction pattern, structure factors, Patterson function to modern methodologies including molecular replacement, SAD, MAD, MIR and maximum likelihood phasing, direct methods, density modification, refinement, model building, twinning and structure validation. Main challenges on sample preparation and crystallization of proteins will also be covered.

The course will be organized with lectures in the mornings and interactive practicals and tutorials in the afternoons. Evening lectures will address two main important topics within Structural Biology, one covering studies of membrane proteins and the other the beginning of the use of free electron lasers in macromolecular Crystallography.

Participants will be limited to 36, aimed primarly at people at the beginning of their crystallographic research activity as PhD students or others. Selected applicants are invited to present a poster during the course.

Speakers and tutors:

Margarida Archer, Isabel Bento, Gabor Bunkoczi, Kevin Cowtan
Zbigniew Dauter, Carlos Frazão, Elspeth Garman, Christoph Hermes
Edward Hough, Gordon Leonard, Andrew Leslie, Bernhard Lohkamp, Adrian Mancuso
Pedro Matias, Rob Meijers, Poul Nissen, Anastassis Perrakis, Célia Romão
Thomas Schneider, Clemens Vonrhein



A registration fee of 600 Euros for academic and 1000 Euros for non-academic applicants is requested for full board and accommodation.
Selected applicants will have to pay the registration fee by bank transfer before arrival.

A limited number of grants is available from FEBS and from IUCr. How to apply

For an application, please fill the form on the web page by the 31th of July.

For more information visit the course web page http://biocrys2012.itqb.unl.pt/

--
Colin E. McVey, DPhil

COOT Real Space Refinement keyboard shortcut??

From: Christopher Browning
Date: 27 March 2012 15:44
Hi,

I was wondering whether there was a way to assign a keyboard shortcut to
the real space refine button in COOT. Atleast this way one does not have
to keep looking back to where the real space refine button is to click
it if you want to carry out the refinement.

Cheers,

Chris




--
Dr. Christopher Browning

----------
From: Debreczeni, Judit
Yes, look here:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot#Example_11:_Paul_Emsley.27s_Key_Bindings

and the key bindings for r, x, t, R.


JED.




>
----------
From: Ed Pozharski
I suspect Chris is asking for the shortcut to the zone refinement
button, i.e. invoking the manual zone selection.  Not sure if there is a
scripting way to do this, nothing obvious.

--
"Hurry up before we all come back to our senses!"
                          Julian, King of Lemurs

----------
From: Petra Lukacik
Dear Chris
Are you trying to use the standard key binding "a" refine with auto-zone?
Mine does not work whilst others such as "m" and "n" for zooming out and in do.

For "a" I get the following message in the terminal:

(graphics-general-key-press-hook 97)
Key 97 not found in (scheme) key bindings

Any ideas on how to fix this, anyone?
I am using Coot 0.6.2 on CentOS 2.6.32-71.29.1.el6.x86_64 x86_64

Regards
Petra

----------
From: Paul Emsley

Are you trying to use the standard key binding "a" refine with auto-zone?
Mine does not work whilst others such as "m" and "n" for zooming out and in do.

For "a" I get the following message in the terminal:

(graphics-general-key-press-hook 97)
Key 97 not found in (scheme) key bindings

Any ideas on how to fix this, anyone?

The activation order is:

1) click "Real Space Refine" icon
2) click atom at in the residue at the centre of range (and centre of screen, typically)
3) press "a" (i.e. the "A" key)

This was built into Coot pre-user-defined-key-bindings.  I rarely, if ever, use it now that there is the "t" short-cut.

In fact, as I write this, I think that I might well take it away so that "A" can be used for something else instead.

Paul.

----------
From: Petra Lukacik
"A" works as described but "t" is better!

In coot 0.6.2 I added the line
(add-key-binding "Triple Refine" "t" (lambda () (manual-refine-residues 1)))

to the file
~/.coot-preferences/coot-preferences.scm

Many thanks
Petra

a question about protein sequences in the PDB

From: Francois Berenger
Date: 27 March 2012 03:54
Dear list,

If I take all the fasta files for proteins in the PDB,
are the sequences complete?

I mean, do they have holes sometimes (missing amino acids)?

Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with the FASTA files.

Regards,
Francois.

----------
From: Bosch, Juergen

I think that depends on what the depositor considered complete.

Just as an example the construct you cloned say from residue 20 - 380 would you consider that complete or would you consider only the sequence complete if it contained the first 20residues ?

Regarding the gaps in terms of missing residues in the structure because they were not observed you shouldn't be worried as they are included in the FASTA sequence.

3TGH just as an example, would you consider that complete ?

Jürgen

......................
Jürgen Bosch

----------
From: Ethan Merritt

On Monday, 26 March 2012, Francois Berenger wrote:
> Dear list,
>
> If I take all the fasta files for proteins in the PDB,
> are the sequences complete?
>
> I mean, do they have holes sometimes (missing amino acids)?

In theory the SEQRES records describe the sequence of the
entity that was crystallized, whether or not it is all visible
in the electron density or present in the deposited model.
So normally there should not be any "missing" internal
residues.  But if the expression construct was a not the full
gene sequence, e.g. an N-terminal truncation, then those
N- or C- terminal residues (or whole domains) will not be
listed.

So goes the theory. There are always corner cases.
I remember having a dispute with the PDB long ago about
whether a peptide chain that was known to have undergone
loop cleavage was properly described with a single
chain identifier or with two chain identifiers.  And if the
cleavage involved excission of one or more residues, would
they appear in the SEQRES records anyhow?

> Sorry for the maybe stupid question but I know that sometimes
> the PDB files have missing residues, I am hoping that
> it is not the case with the FASTA files.

I was assuming that the FASTA files you refer to are just
conversions of the SEQRES records.  If not, then all bets are
off.  If the FASTA files are retrieved by gene ID from Uniprot
or some other sequence data base, then they will be complete in
one sense but may not perfectly match what was in the deposited
crystal structure due to cloning artifacts, strain variation,
allelic non-uniformity, etc.

       Ethan

> Regards,
> Francois.
>

----------
From: Francois Berenger

OK, thanks for the answers.
I'll try to find out more about the FASTA files present in the database then.

Regards,
F.

       Ethan

Regards,
Francois.

----------
From: Chad Simmons

The total model that fits the observable electron density should be the standard for the PDB FASTA file with the deposited structure factors, however, not all depositions contain a link to the expression construct details because many are not published, and I believe that it should be explicitly detailed in the submission.

Chad

granular precipitate

From: anita p
Date: 27 March 2012 16:02

Hi All,
I have set up initial screen in hanging drop trays with a protein of theoritical pI of 8.5. The protein is in acetate buffer 10mM, KCl 100mM and 2% glycerol
pH 5 . In 85-90% of the conditions I see granular precipitate in 1 day. I tried to open the coverslip, and touch few drops, They had a skin layer and the granular looking thing are part of the skin. The protein was set up at 10 and 5 mg/ml concentration. Is this normal?

I have tried to change the protein buffer to Hepes at pH 7 with other constituents same,and tried to set up few screen trays. I cant see granular precipitate again. The precipitates look light brown and amorphous in nature.

Is Na acetate forming some granular precipitate instead of protein?

Kindly suggest me ways to move forwards.
Thanks in advance
Anita

DDM

From: Katarzyna Rudzka
Date: 26 March 2012 18:17

Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb.  I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow

----------
From: yybbll

Hi,

I used 0.017% or 0.012%. My protein is very stable at this concentration.

Good luck.

Lin

2012-03-26

---

yybbll

---

----------
From: Kelly Daughtry
I generally use 1 - 2% DDM for extraction only, but lower the concentration to 0.01% for following steps (i.e. NiNTA and gel filtration). The excess DDM is washed away by using a lower concentration in your wash and elution buffers.

Kelly

----------
From: Das, Debanu
Hi Katarzyna,

Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value is very low, at the extraction step you need to use a much higher concentration up to ~100x CMC. During different rounds of purification, you can bring the CMC level down to 1-2x CMC and even try detergent exchange in the last step, although it can be difficult to totally exchange out the DDM due to its low CMC. Also use a 100 kDa MWCO concentrator since the DDM micelle size is large.

In general, DDM is a reasonable choice for extraction, purification and crystallization setups, but it will be worthwhile to screen a panel of different detergents for extraction and purification for your particular target.
In 2005, we set up a fast protocol for screening a panel of 18 detergents in 48-72 hours, http://smb.slac.stanford.edu/~debanu/posters/012605_PPCW2005_DDAS.pdf, which includes suggestions for starting concentrations for extraction.

There are also some papers on detergent screening, for example:
http://www.ncbi.nlm.nih.gov/pubmed/18988031
http://www.nature.com/nprot/journal/v4/n5/full/nprot.2009.27.html (Stroud lab)

Thanks,
Debanu.

----------
From: Joao Dias

Kasia,

A lot of people uses DDM to purify membrane proteins, not a lot of people crystallises them.

If you want to crystallise a protein purified in DDM, then you should use LCP.

If you go for vapour diffusion, you should exchange the DDM for a detergent with a smaller micelle size otherwise you might get crystals but it is difficult to get good diffraction. Try mixed micelles for example.

Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in order to prevent detergent concentration.

For extraction it depends on your protein and expression system but you can see in the literature values between 0.5-2% being used successfully.

Good luck.

Cheers,

Joao

Joao Dias, Ph.D.

----------
From: Edward A. Berry
If I recall correctly cytochrome oxidase, which I believe was
the first protein purified with DDM, requires about 10x cmc
in column buffers to keep it soluble. Check for papers from 1970's or 80's
by S. Ferguson-Miller.

Cytochrme bc1 complex, on the other hand, is perfectly clear in 1 cmc
and can be diluted from that into detergent-free buffer for
spectroscopywithout becoming turbid, at least within an huor.

My rule of thumb for solubilizing with DDM is 1 g/g protein (+ 1 cmc).
If protein is say 10-20 g./l, the (+ 1 cmc) becomes completely insignificant.
(Total protein, not the protein of interest. Probably depends on the
amount of lipid too, but this is just a rule of thumb to start with.)

eab

----------
From: Ho Leung Ng
Actually, DDM is the most successfully used detergent for membrane
protein crystallization. See Newstead et al, Protein Sci. 17:466. But
yes, the rule of thumb is that detergents that form smaller micelles
give better diffracting crystals, but are more destabilizing.


Ho

Ho Leung Ng

----------
From: Daniel Picot
It is important to distinguish between the solubilisation and the purification steps:
1) During the solubisation step you need to care about the lipid/detergent ratio. The amount (not the concentration) of detergent (i.e. in non monomeric form, the detergent above the cmc) is important. You may need a high amount of detergent.
2) During the purification step, you need to keep your protein soluble. Here, the concentration is important and you may  keep the concentration of detergent around the cmc. But you have to be aware that in the initial (and also the not so initial ones!) steps you may have a lot of lipids, you need then keep the concentration of detergent fairly high in order to keep everything soluble. I like to decrease the detergent concentration at each purification steps in order to avoid protein denaturation. The protein may sustain a fairly high detergent concentration of detergent during the early steps since the lipid/detergent mixed micelles will be less denaturing than the pure detergent micelles in the later steps. Thin layer chromatography is a quick and easy method to check if you have a large amount of lipids in your preparation.
HTH
Daniel

change in unit cell parameters and difficult MR

From: intekhab alam
Date: 27 February 2012 06:44

Hi all

I have collected 2 datasets; one for the native proteins (2.3Å resolution) while other is for a protein-protein complex (3.5Å resolution). The unit cell parameters for the native are a=120, b=196, c=109, a=g=90, b=114, while for the complex data it is a=122, b=197, c=300, a=g=90, b=93. I have successfully got a MR solution and refined the native dataset with 3 monomers in the asymmetric unit. Analysis of the protein complex dataset shows 41% pseudo translation and so far there is no success in molecular replacement for this dataset. Can anyone please suggest me ways to solve this problem. i already have tried different templates like monomer, dimer, trimer, tetramer etc with truncations of loop region. i am using phaser as well as molrep programs.

Regards
--
INTEKHAB ALAM

----------
From: Eleanor Dodson


 Well, there is an obvious relation between cell dimensions - what is the translation vector you find at 41%

You say the native structure has 3 monomers - I would send those coordinates to the PISA application at the EBI, and see what the interfaces are and make sure you are using closely packed search models - eg you may find there is a closely packed trimer which is built using symmetry equivalent molecules to your native solution..

Hard to answer without more information though.
ie space group, relative size od protein and complex etc..
Eleanor

Professor Eleanor Dodson

Heterotetrameric protein

From: Koustav Maity
Date: 26 February 2012 00:26

Hi all,

  does anyone know a protein which make hetero tetramer of A2B2 which arrange as  and NOT as ?
                                                                                                                                              
Its great if these proteins are small? All the hetero tetramer I found was of  type.
    

Thanks

Dhiraj

----------
From: Tanner, John J.


Tryptophan synthase forms a ABBA tetramer (PDB 2J9X).  It is not exactly what you want, but it is different from what you have found.

Jack Tanner

John J. Tanner

----------
From: Lavy, Tali


Hi Dhiraj,

We've just submitted such a heterotetramer to the pdb (3V2U)

Tali








----------
From: Eleanor Dodson


All haemoglobins do..
Eleanor

--
Professor Eleanor Dodson

CCP4i Reflection DataUtilities: Convert to/modify/extend MTZ does not work

From: Dialing Pretty
Date: 29 February 2012 01:58

Dear All,

I want to use "CCP4i Reflection DataUtilities: Convert to/modify/extend MTZ" to change a RCSB mmCIF file into MTZ file.

It shows there are a lot of data name (related to _pdbx_.or something else) not present in dictionary, and shows CCP4I termination status 0 "child process excited anormally".

I am looking forward to getting a reply from you on how to solve the problem.

Cheers,

Dialing

 

----------
From: Jayashankar

I face exactly the same problem. any advice in this regard would be greatful.


S.Jayashankar 

----------
From: Martyn Winn


If you send me the PDB id that you are trying to convert, I can
investigate.

This issue has come up a few times recently on the BB - you can also try
searching the archives for advice.

The version of cif2mtz + dictionary in the forthcoming 6.3 should cope
with a wider range of cif files.

Cheers
Martyn

--

**********************

----------
From: Dialing Pretty

I just have it successfully done: 

Import reflection file in mmCIF format and create MTZ file

In 1w2i-sf.cif

----------
From: Martyn Winn


Yep, works for me too.

1w2i-sf.cif contains no pdbx entries. Did you perhaps first download the
mmcif format coordinate file?

On the RCSB site, the "mmCIF File" are coordinates, and "Structure
Factor (text)" are reflections in mmCIF format.

Cheers
Martyn

Postdoctoral position, Institut de Biologie Stucturale, Grenoble, France

From: Franck Borel
Date: 22 March 2012 15:29

Postdoctoral position, Institut de Biologie Stucturale, Grenoble, France

A two year postdoctoral position for a biochemist/structural biologist is available in the Synchrotron Group at the Institute for Structural Biology (IBS) in Grenoble (France).
The IBS has state-of-the-art equipments and facilities, including crystallization robots, NMR, EM and easy access to synchrotron beamlines. The subject is focused on the structural analysis of a CDK/Cyclin complex, in a drug design approach. The candidates should hold a Ph.D. in biochemistry or biophysics. A solid experience in protein expression, purification and biochemical characterization is required. Knowledge and experience in protein crystallography and in in silico docking will be an advantage. The candidates must be motivated, well-organized and able to work independently as well as a part of the team.
Salary is in the range 30-35 k€, depending on experience. The position is immediately available and interested candidates should send their CV to franck.borel @ ibs.fr

recommendations_on_purification

From: Petros Giastas
Date: 26 March 2012 13:04

Dear all,

I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions?

Thanks in advance
Petros

----------
From: Lorenzo Finci

Petros, 

It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of . Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.

Sincerely, 

lorenzo

Lorenzo Ihsan FInci, Ph.D.

----------
From: Lorenzo Finci

Etros, 

It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of Imidazole your protein elutes at by using a gradient before progressing to a step-wise elution. Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.

Sincerely, 

lorenzo

Lorenzo Ihsan FInci, Ph.D.

----------
From: Carlos Kikuti

90% of the protein could be aggregated and hiding the His tag from the resin. Metal ions might be removed from the starting sample by adding up to 10 mM EDTA in the first buffer exchange cycle, but just after reading "10% of the protein binds" I wouldn't bet much on this. Rather increase the culture volume and move on.

Carlos Kikuti

----------
From: Artem Evdokimov
Hi Petros,

It's possible but unlikely that your media contains enough metal ions
to matter. More likely options include protein issues like
over glycosylation, proteolysis (removal of the tag) or severe
aggregation. Since you are not experiencing this in BMGY, I wonder why
not to supplement your synthetic medium with yeast extract and/or
peptone and the like?

Artem

----------
From: Kendall Nettles

I suspect that sometimes the protein chaperones the tag, which is solvent exposed some fraction  of the time.  Try very slow loading or batch binding. 

Kendall Nettles