Re: [ccp4bb] over-expression strategies

2013-09-13 Thread Phoebe A. Rice 
Something I recently learned from a friend: try changing the 4th base in the 
coding sequence to G (e.g. ATGGxx...) and the ribosome will like it better.  We 
tried it recently on a frustrating construct and it worked like a charm.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Fernandez, Elias 
J [efern...@utk.edu]
Sent: Friday, September 13, 2013 7:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] over-expression strategies


Yes, the gene's been codon-optimized. The predicted mRNA structure appears 
stronger in the optimized version than the native, where it is high too. And, 
expression levels are unchanged. Elias


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Elias Fernandez 
[efern...@utk.edu]
Sent: Thursday, September 12, 2013 10:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] over-expression strategies

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

Re: [ccp4bb] Adding ligand to crystallization drop

2013-09-13 Thread Roessler, Christian 
Another thing to try if you want to "speed up" the soaking process is to 
optimize your crystal growth to give smaller crystals.  10-20 micron crystals 
can reach 80% occupancy in less than 30 seconds, at least in our very limited 
tests.  But this quick soak method was important for our system as exposures 
over 1 minute in the DMSO-solvated compounds killed the crystals.

Good luck!

Chris



---
Christian Roessler, PhD
Structural Biology Group
Brookhaven National Laboratory
Upton, NY 11973
croess...@bnl.gov
(631) 344-7621



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Christopher Browning
Sent: Thursday, September 12, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Adding ligand to crystallization drop

Thanks for the really helpful tips. Right now I've tried 5, 10 and 16% DMSO 
(due to variations in the final ligand conc.) and the crystals visually look 
happy. I'll definitely give the cryo/ligand mix method a go as this seems to do 
the job all at once. I already know what cryo to use.

Thanks,

Chris



--
Christopher Browning, Ph. D

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

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

From: , Juergen mailto:jubo...@jhsph.edu>>
Date: Thursday, 12 September 2013 15:09
To: Christopher Browning 
mailto:christopher_b_brown...@vrtx.com>>
Cc: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Adding ligand to crystallization drop

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

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

Jürgen

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

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


Hi Again,

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

Cheers

C



--
Christopher Browning, Ph. D

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

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

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

Chris,

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

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

with that being said, soaking can sometimes lead to partial occupancy of your 
ligand.  So if you are able to do co-xtallization, i think that would be 
preferred and doesn't seem like too much additional effort assuming you are 
getting crystals in the same conditions as with native protein only.  and with 
co-xtallization, I often see slightly better resolution that with native 
protein only (not always the same condition though unfortunately

Re: [ccp4bb] weekend puzzle

2013-09-13 Thread Paul Emsley 

On 13/09/13 17:50, Andreas Förster wrote:

Dear all,

a structure I'm refining is enjoying a free R factor of 18 but still 
suffers from mystery density that nothing in the crystallization 
condition could explain.  I can't explain it either.  Maybe you like 
to venture some guesses?


The missing ligand is derivatized benzoic acid, nitrobenzene or 
benzamide.  I didn't find anything obvious in the PDB.  Any suggestions?

http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle1.png

The second ligand is a longer shot, two blobs connected by a 
substantial bridge.  The two views are slightly rotated, and a bit of 
peptide is shown to give an idea of the size.

http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle2.png
http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle3.png

Happy guessing!


This is what I can detect from your images...

it's not really a nugget, is it? It's more of a splat.

(it *is* Friday afternoon, that's my excuse...:))

Paul.

<>

[ccp4bb] weekend puzzle

2013-09-13 Thread Andreas Förster 

Dear all,

a structure I'm refining is enjoying a free R factor of 18 but still 
suffers from mystery density that nothing in the crystallization 
condition could explain.  I can't explain it either.  Maybe you like to 
venture some guesses?


The missing ligand is derivatized benzoic acid, nitrobenzene or 
benzamide.  I didn't find anything obvious in the PDB.  Any suggestions?

http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle1.png

The second ligand is a longer shot, two blobs connected by a substantial 
bridge.  The two views are slightly rotated, and a bit of peptide is 
shown to give an idea of the size.

http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle2.png
http://www.msf.bio.ic.ac.uk/images/puzzle/puzzle3.png

Happy guessing!


Andreas




--
  Andreas Förster
 Crystallization and Xray Facility Manager
   Centre for Structural Biology
  Imperial College London

[ccp4bb] postdoctoral position in crystallography

2013-09-13 Thread Bing Chen 
A postdoctoral position in x-ray crystallography is available 
immediately in Bing Chen’s laboratory at Boston Children’s 
Hospital/Harvard Medical School. A strong background in protein 
crystallography is required for working on challenging crystals of viral 
envelope glycoproteins. Previous experience in solving difficult 
crystallographic problems is preferred. We are particularly interested 
in candidates who are highly motivated and willing to tackle difficult 
problems. Applicants should send a cover letter, CV, and contact 
information of three references to Bing Chen at bc...@crystal.harvard.edu.

Re: [ccp4bb] video of crystal pick-up through microscope?

2013-09-13 Thread Swastik Phulera 
Hi,
May be this would help
https://www.youtube.com/watch?v=QcsaWowulDM

On Fri, Sep 13, 2013 at 2:51 PM, Frank von Delft
 wrote:
> Hi all - does anybody know where I can find a video of a crystal being
> harvested, but taken through a microscope?
>
> The Google has not been helpful, but I'm obviouly incompetent with these
> new-fangled things.
>
> Thanks!
> phx



-- 
--
स्वस्तिक फुलेरा
वरिष्ठ अनुसंधान कर्ता
संरचनात्मक जीवविज्ञान प्रयोगशाला
डी. एन. ए. फिंगरप्रिंटिंग एवं निदान केंद्र
हैदराबाद, ५००, ००१

और

राष्ट्रीय कोशिका विज्ञान केंद्र
पुणे विश्वविद्यालय परिसर,
गणेशखिंड
पुना,  ४११,००७

Re: [ccp4bb] over-expression strategies

2013-09-13 Thread Fernandez, Elias J 

Yes, the gene's been codon-optimized. The predicted mRNA structure appears 
stronger in the optimized version than the native, where it is high too. And, 
expression levels are unchanged. Elias


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Elias Fernandez 
[efern...@utk.edu]
Sent: Thursday, September 12, 2013 10:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] over-expression strategies

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

[ccp4bb] video of crystal pick-up through microscope?

2013-09-13 Thread Frank von Delft 
Hi all - does anybody know where I can find a video of a crystal being 
harvested, but taken through a microscope?


The Google has not been helpful, but I'm obviouly incompetent with these 
new-fangled things.


Thanks!
phx

[ccp4bb] Registration last day: "The Future of Microfocus Protein Crystallography"

2013-09-13 Thread Marjolein Thunnissen 
Dear all,
I would like to remind you on a workshop on "The Future of Microfocus Protein 
Crystallography" which will take place at the MAX IV User meeting on the 24th 
and the 25th of September, 2013, in Lund, Sweden.

Recent developments within protein crystallography methods in combination with 
the scope offered by the new upcoming synchrotron sources such as the MAX IV 3 
GeV storage ring, make exciting new ways of carrying out research within 
structural biology possible. Small microfocus beamlines with a very high flux 
and increased coherence will make new types of experimentation feasible.

The scientific possibilities will be highlighted through a series of talks by 
experts showing the impact of the new type of X-ray sources and microfocus 
beamlines on research within structural biology.

The new generation of microfocus beamlines will also set a high demand on 
instrumentation and suitable software. For example, data collection will 
potentially be very fast (in milliseconds) and radiation damage will allow only 
a limited amount of data to be collected from each sample. New technical 
solutions for sample handling and presentation, data processing methods and 
more will be needed for these types of experiments, and these will also be 
presented and discussed at the workshop.

Confirmed speakers include David Stuart, Daniel Wacker, Simone Weynand, Jens 
Preben Morth, Tove Sjögren, Richard Neutze, Janet Smith, Alke Meentz, Alexei 
Soares, Thomas White, Luca Jovine and Clemens Schulze-Briese

The workshop is part of a process to obtain an updated proposal for the 
possible Phase 2b beam line MicroMAX at the upcoming new 3 GeV storage ring of 
MAX IV in Lund. This workshop should give the user community an opportunity to 
explore the scientific possibilities of a new generation of microfocus beam 
lines, but also it will give them the chance to contribute with specific input 
on their own experimental demands on the beam line.

The workshop is part of the MAX IV laboratory user meeting, but there is no 
requirement to be a user of the facility and all that are interested are 
welcome . Registration is through the user meeting website: 
https://indico.maxlab.lu.se/indico/conferenceDisplay.py?confId=0 .  The 
registration fees are 2000 SEK for academics and 700 SEK for junior scientists 
such as PhD students and Post-docs. Registration fees include all lunches and 
the conference dinner.

Further information on this workshop can be found at the user meeting 
websiteand for the MAX IV user meeting at https://www.maxlab.lu.se/um13 .

For the organisers, Richard Neutze, Gunter Schneider and Gregers Rom Andersen

Marjolein Thunnissen


 Marjolein Thunnissen Phone +46-(0)76-632 0417
 Associate Professor  Fax   +46-(0)46-22 24692
 Dept of Biochemistry and Structural Biology, Lund University
http://www.mps.lu.se
 PO-Box 124 S-221 00 Lund, Sweden

Scientific coordinator MX (The MAX IV Laboratory): I911 and BioMAX

[ccp4bb] Postdoc in Pearl Lab on Hsp90 Complexes - correction

2013-09-13 Thread Laurence Pearl 
Postdoctoral Research Fellow in Structural Biology of Hsp90 Complexes (Fixed 
term) Ref 297

School of Life Sciences - Genome Damage and Stability Centre

Full time, fixed term for 36 months
Salary range: starting at £30,424 and rising to £36,298 per annum.
Closing date for applications: 27 September 2013

Description

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

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

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

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

Ref 297 Further particulars including person specification [PDF 
78.54KB]

Essential information
Terms and conditions

Research Faculty Terms and Conditions SUMMARY [DOC 
36.00KB]

Application

Application form - academic post [DOC 
301.50KB]
Application form - academic post [PDF 
57.04KB]

Please send completed application form by email 
to:lifescirecruitm...@sussex.ac.uk

When emailing your application please use the following format in the 'subject' 
line: (post reference number / post title / your name). If you are applying for 
more than one post advertised by the University, please send a separate email 
and application form for each post. Please attach your completed application 
form and any other documents directly to the email rather than using a 
web-based upload / weblink service (e.g. SkyDrive) otherwise we may not receive 
your application due to incompatibility with our email software.

Or post to: Human Resources Division, Sussex House, University of Sussex, 
Falmer, Brighton, BN1 9RH.

Applications should be received by midnight on the closing date.


Laurence H. Pearl PhD FRS FMedSci

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

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

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