[ccp4bb] PhD position at the Membrane Protein Laboratory (Diamond)

[Dr. Isabel De Moraes]

Applications are invited for a 3-year PhD position at Imperial College London 
which will be based in The Membrane Protein Laboratory (MPL) at Diamond Light 
Source, Didcot, Oxfordshire and the Centre for Structural Biology, Imperial 
College London. The successful applicant, for the position which is immediately 
available, will work on structural studies of membrane transport proteins using 
microfocus X-rays and X-ray Free Electron Laser beams.  The project “NanoMem” 
is supported by the Marie Curie Initial Training Network (European Commission 
Framework Seven Programme).
The MPL operates both as a facility for users and as a cutting edge research 
laboratory. It is a high-throughput protein crystallisation facility that aims 
to enhance the rate of success in the crystallisation of medically significant 
membrane proteins. Its location at Diamond Light Source facilitates 
high-throughput crystal screening and high quality data collection.

The student will be co-supervised by Prof. So Iwata, Dr Isabel Moraes and Prof. 
Xiaodong Zhang at Imperial College. The student will be trained in 
state-of-the-art methods of membrane protein expression, purification, 
crystallization and structure determination using both synchrotron microfocus 
X-Ray beamlines and X-Ray free electron lasers.  The successful candidate, as a 
member of the Marie Curie Initial Training Network, will participate in 
collaborative experiments, secondments and training events across the network 
(The NanoMem network compromises ten laboratories from five European countries).

Studentship Details
Applicants should hold a Masters degree in Biochemistry or a related subject.
To apply: applications should include a cover letter describing relevant 
research experience to date, a CV, and the names and addresses of two referees.
These should be sent to Dr Isabel Moraes 
(i.mor...@imperial.ac.uk) by email by 31th May 
2013.
This studentship is only available to applicants who are not UK citizens or 
have not lived in the UK for more than 12 months during the last three years.
For informal enquires please contact Dr Isabel Moraes  
(i.mor...@imperial.ac.uk)




Dr Isabel De Moraes, MRSC

Head of the Membrane Protein Laboratory

Diamond Light Source Ltd,
Harwell Science and Innovation Campus,
Chilton, Didcot, Oxfordshire,
OX11 ODE, UK






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[ccp4bb] Scaling problem with multiple crystals

[amit sharma]

Dear All,

I have been trying to scale (using SCALA) the diffraction data set collected 
from multiple crystals. Data was indexed using mosflm but fails to scale with 
an error saying 
"negative scale factors" when scaling was tried in a constant mode whereas it 
fails to scale with an error saying "No observations" when scaling was tried in 
a batch mode. Damping shifts were tried from 0.1 to 0.5 and also unit 
weighting, but the scaling still fails with same errors. Can someone provide 
any suggessions to deal with the problem.

Kind Regards,

Amit

Re: [ccp4bb] Scaling problem with multiple crystals

[Phil Evans]

Depending on the Space (Laue) group, there may be alternative indexing 
possibilities which will be sorted out in Pointless - did you run that program?

I would recommend using the ccp4i task "Symmetry, Scale, Merge (Aimless)" which 
uses Aimless instead of Scala - Aimless may be a bit more robust than Scala in 
some cases

Other things to try.
 If you have several crystals, one (or more) may just be "bad". You can try 
scaling them separately, or using the "scale constant" option just to diagnose 
which crystals would be better omitted

Phil


On 10 May 2013, at 16:04, amit sharma  wrote:

> Dear All,
> 
> I have been trying to scale (using SCALA) the diffraction data set collected 
> from multiple crystals. Data was indexed using mosflm but fails to scale with 
> an error saying 
> "negative scale factors" when scaling was tried in a constant mode whereas it 
> fails to scale with an error saying "No observations" when scaling was tried 
> in a batch mode. Damping shifts were tried from 0.1 to 0.5 and also unit 
> weighting, but the scaling still fails with same errors. Can someone provide 
> any suggessions to deal with the problem.
> 
> Kind Regards,
> 
> Amit

Re: [ccp4bb] Membrane Protein Optimisation

[John Lee]

Hi Rhys,

The bOG concentration of 1.0% is quite acceptable and if your protein is
stable and happy in OG, then you're in luck. At 30% PEG 600, I'm not
surprised you're seeing the phase separation or detergent 'blob's for lack
of a better term. I believe you have the latter, which many of us working
with memb proteins experience.

You are probably at above 1% OG though. When you concentrate, do you use
100kDa cutoff membrane. Based on the size of your protein, this would be a
safe concentrator to use. FYI, I have several proteins that are ~50kDa, and
I can use 100kDa concentrators without ill effects.

If you are using 100kDa membrane, then you might try 'concentrating' via an
ion-exchange column before actual concentration.

Lastly, as Jim mentioned, you might want to change your construct by a few
residues. This might help with the resolution.

-john


On Thu, May 9, 2013 at 3:49 PM, RHYS GRINTER  wrote:

> Thanks for the suggestions so far, I should have given a little more
> information in my initial post. The protein I'm working on is an Gram-Neg
> Beta-Barrel about 100kDa, likely with 22 strands.
>
> I'm currently crystallising in bOG, although my next optimisation step is
> to try a range of detergents.  I'm using a refolding protocol which relies
> heavily in LDAO (one of the only cost effective detergents for the volumes
> I'm using), I refold, nickel purify (N-terminal tag, signal peptide
> removed) , do a size exclusion step (S200), then detergent exchange into
> bOG using a nickel column. The BOG concentration is obviously high 0.8-1%,
> which might be causing the gels?
>
> The gels don't look like classic phase separation to me (from soluble
> proteins that is, this is my first membrane protein), they are not
> spherical, the tend to float or sit on the bottom and are semi-solid,
> around 30-100uM diameter. These screens I've set up are classic screens for
> membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't
> dried up. The gels are strongly birefringent, but the drop is not.
> Additionally the conditions in my initial screen which yielded crystals
> seemed biased for the formation of the gels.
>
> Thanks again for the help, I feel I might have a long raod to optimisation
> ahead of me.
>
>
> Rhys
>
> 
> From: Jim Fairman [fairman@gmail.com]
> Sent: 09 May 2013 20:58
> To: RHYS GRINTER
> Cc: ccp4bb
> Subject: Re: [ccp4bb] Membrane Protein Optimisation
>
> With information you've provided I have multiple suggestions for you, some
> of which you may have already tried:
>
> 1.   OMPs can be fairly particular about which detergents they will
> crystallize in.  Try exchanging the protein into a different
> detergent/detergent mixture and then set up new trays in your favorite
> broad matrix screens.  DDM, DM, LDAO, OG, and C8E4 are a few of my
> favorites for OMPs, but there are many others.  This can be done using a
> size exclusion column as the final purification step for your protein where
> the column is equilibrated with the appropriate detergent.  This step will
> also let you know how well behaved the protein is in that detergent via the
> shape/height of the peak.  This won't help you optimize your current
> condition, but it may lead to different/new conditions with even better
> crystals.  As Pascal suggested, try to carefully choose which MW cut-off
> concentrator you end up using.  Minimizing the amount of detergent
> concentration that occurs during your concentration step(s) is optimal.
>
> 2.  Attempt crystallization using DHCP/CHAPSO bicelles.  You can buy them
> pre-made from MemX Biosciences or make them yourself using a published
> protocol from David Bowie's lab.  These have been used to crystallize the
> mitochondrial beta barrel protein VDAC and I had success crystallizing
> intimin in them.  David Bowie also has a JoVE article on the bicelle
> crystallization method.
>
> 3.  Attempt crystallization using Lipidic Cubic Phases.  This was the
> saving grace for my post-doc project.  Neither detergent nor bicelle
> crystallization produced crystals that were of sufficient quality, but the
> crystals from LCP all diffracted to the 2.0 angstrom resolution range.  If
> you're unfamiliar with the technique, there are several nice videos on JoVE
> describing it by Vadim Cherezov and Martin Caffrey.
>
> 4.  Alter your construct.  Small changes in the construct (ie: deletion or
> addition of 1-2 residues on the N- or C-terminus) often led to drastically
> different crystallization behavior of several OMPs in my hands.
>
> Cheers, Jim
>
>
> On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER <
> r.grinte...@research.gla.ac.uk>
> wrote:
> Hi All,
>
> A quick question if you've ever worked on membrane proteins, I'm trying to
> optimize crystals for bacterial integral outer membrane protein I'm working
> on. I'm getting some fairly modest rod like crystals in a  0.1M Tris pH
> 7.5, 30% PEG 600, 

Re: [ccp4bb] Membrane Protein Optimisation

[Ho Leung Ng]

Hello Rhys,

 I suggest using an assay such as that described in Anal. Biochem.
336:117 to measure the amount detergent in your concentrated samples.
I found this assay accurate and easy to use with DDM. It should work
with b-OG too.


Good luck,
Ho

Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
h...@hawaii.edu

[ccp4bb] 43rd Mid-Atlantic Macromolecular Crystallography Meeting

[Charles Pemble]

*43**rd** Mid-Atlantic Macromolecular Crystallography Meeting*

*May 30 – June 1, 2013*

*Duke University*

*Durham, NC*




*ANNOUNCEMENT:  *We have extended the deadline for submitting abstracts to
be considered for presentations until midnight Sunday, May 12.  Registration
and details can be found at http://www.mid-atlantic.org.


The 43rd Mid-Atlantic Macromolecular Crystallography Meeting, will be held
at Duke University in Durham, North Carolina.  This year will feature
keynote speaker Tom Terwilliger from Los Alamos National Lab, workshops on
PHENIX and MolProbity, plus many talks & posters on new and exciting
research.  The best presentation and poster by a Graduate Student &
Postdoctoral Fellow will be awarded (total of 4).  We encourage you to
submit your abstracts and sign up for the workshops as these are separate
processes from registration.



*DEADLINES (updated):*

Abstract submission for presentation - May 12th

Abstract submission for poster - May 27th

*No deadline for registration







With best wishes on behalf of the primary Organizing Committee,



Charlie Pemble

_

Charles W. Pemble IV, Ph.D.

Duke University Medical Center

308 Research Drive

LSRC, A06

Durham, NC 27708

charles.pem...@duke.edu



We thank our current sponsors for their generous contributions:  Rigaku
Americas Corporation, Agilent Technologies Inc., GlaxoSmithKline, Art
Robbins, Bruker, HKL Research Inc., Molecular Dimensions, Hampton Research,
Labcyte, GenScript, Genentech, Emerald BioSystems, TTP LabTech, Oxford
Cryosystems, PhD Posters, Duke University School of Medicine, Duke Human
Vaccine Institute, and Avanti Polar Lipids Inc.