[ccp4bb] Research associate position available in structural biology at Imperial College

[Wilkinson, Martin]

Dear all,


Please see attachment for a structural biology job advert in the lab of 
Professor Dale Wigley at Imperial College.


Closing date for applications is 10th March.


For informal enquiries please contact Professor Dale Wigley 
(d.wig...@imperial.ac.uk)


Cheers, Martin




HM2017001 Research Associate Advert QC SW - extended.pdf
Description: HM2017001 Research Associate Advert QC SW - extended.pdf

Re: [ccp4bb] Estimating the amount of missing electron density for a model

[Ethan A Merritt]

Thank you Randy and Pavel for correcting my failure to find an
appropriate tool in my mental toolbox.  I was thinking too much
in terms of examining a difference map rather than a performing
a resolution-dependent analysis of Fo;Fc agreement.

Is there a straight-forward way to extract what Hunter wants from
the output of SIGMAA run from the ccp4i interface?

Ethan

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] Estimating the amount of missing electron density for a model

[Hunter Moseley]

Randy,
Use of correlation makes normalization superfluous, but as you say it
cannot distinguish from the causes of deviation which may not just be
missing density.

Pavel,
Thanks for the reference. Will look at it more closely.
But how is the error being separated into separate error components alpha
and beta?

Cheers,
Hunter

On Wed, Feb 22, 2017 at 11:28 AM, Pavel Afonine  wrote:

> Hi,
>
> Is there a straight-forward way to estimate the amount of missing electron
>> density that a particular protein structure is missing based on the
>> difference between Fo and Fc?
>>
>
> Any refinement or map calculation software that uses likelihood-based
> approach does this routinely. In phenix.refine and phenix.maps I coded this
> approach : Acta Cryst. (2002). A58, 270-282. Here it calculates alpha/beta
> parameters by comparing Fo and Fc taken from free set. alpha is a function
> of model error, and beta is proportional to amount of missing scattering.
> If you look up the formulas it should be possible to convert alpha into
> error in coordinates (in A) and beta into number of missing atoms (of some
> general type). ML based estimate of error in model that phenix.refine
> reports is actually done this way.
>
> Pavel
>
>


-- 
Hunter Moseley, Ph.D. -- Univ. of Kentucky
Associate Professor, Dept. of Molec. & Cell. Biochemistry / Markey Cancer
Center
/ Resource Center for Stable Isotope Resolved Metabolomics
 Not just a scientist, but a fencer as well.
   My foil is sharp, but my mind sharper still.
---
Email: hunter.mose...@uky.edu (work)   hunter.mose...@gmail.com
(personal)
Phone: 859-218-2964 (office)   859-218-2965 (lab)   859-257-7715 (fax)
Web: http://bioinformatics.cesb.uky.edu/
Address: CC434 Roach Building, 800 Rose Street, Lexington, KY 40536-0093

Re: [ccp4bb] Estimating the amount of missing electron density for a model

[Pavel Afonine]

Hi,

Is there a straight-forward way to estimate the amount of missing electron
> density that a particular protein structure is missing based on the
> difference between Fo and Fc?
>

Any refinement or map calculation software that uses likelihood-based
approach does this routinely. In phenix.refine and phenix.maps I coded this
approach : Acta Cryst. (2002). A58, 270-282. Here it calculates alpha/beta
parameters by comparing Fo and Fc taken from free set. alpha is a function
of model error, and beta is proportional to amount of missing scattering.
If you look up the formulas it should be possible to convert alpha into
error in coordinates (in A) and beta into number of missing atoms (of some
general type). ML based estimate of error in model that phenix.refine
reports is actually done this way.

Pavel

Re: [ccp4bb] Estimating the amount of missing electron density for a model

[Randy Read]

Hi,

I rarely disagree with Ethan, but there are in fact ways of getting some idea 
of how much of the ordered structure from your crystal is missing in your 
model.  It's not based on the difference between Fo and Fc but rather more on 
the correlation between them and how that varies as a function of resolution.  
How well this works depends very much on why your model is incomplete — in many 
circumstances Ethan will be right in practice!

Some time ago, I proposed the use of something called a SigmaA plot 
(http://www-structmed.cimr.cam.ac.uk/Personal/randy/pubs/a25349r.pdf).  SigmaA 
is a parameter that measures what fraction of the calculated structure factor 
is correlated to the observed structure factor in a particular resolution 
shell.  The correlation depends on both the fraction of the crystal that has 
been modelled and on the quality of the model.  Since the effect of errors in 
the model is greater at higher resolution, SigmaA is a function of resolution.  
The way it varies with resolution tells us something about the size of the 
errors in the model, and the limiting value at low resolution tells us 
something about the completeness of the model.  If we make some assumptions 
(most importantly that the errors are primarily coordinate errors that are 
drawn from the same isotropic 3D Gaussian distribution of errors for all atoms, 
and that the missing part of the structure has the same overall B-factor as the 
modelled part of the structure), then we expect a plot of the logarithm of 
SigmaA against 1/d^2 to be a straight line, where the slope is proportional to 
the mean-square coordinate error and the intercept is proportional to the 
logarithm of the model completeness.

By the way, the original SigmaA plot also assumed that the measurement errors 
in the diffraction data were negligible.  With the development of our new LLGI 
(intensity-based likelihood) target, we now have a way of correcting the 
estimation of SigmaA for the effect of errors, so we could do better if there 
was a resurgence of the SigmaA plot.  

We don't model bulk solvent nearly as well as ordered atoms, so these plots are 
not very linear at low resolution.  But the plots do tend to be surprisingly 
linear at higher than about 5-6A resolution.  If you're doing molecular 
replacement on a complex and you place a model for part of the complex, the 
intercept of a SigmaA plot computed before refining your model will usually 
give you a reasonable idea of how much is still missing.  Refining the model 
could make things worse because of the effects of overfitting your data.

However, the case of a complex was assuming that the missing part is as well 
ordered as the modelled part.  Frequently we have trouble completing a model 
because a domain or an NCS copy has much higher overall B-factors.  What that 
means is that the fraction of the scattering we can model also varies with 
resolution instead of being a constant, because the better ordered part 
accounts for an increasing fraction of the scattering at high resolution.  This 
violates the assumption about the coordinate error being the only thing that 
makes SigmaA vary with resolution.

So whether this might be useful to you will depend on where you are with the 
structure determination and why your model is incomplete!

Best wishes,

Randy Read

> On 21 Feb 2017, at 21:53, Hunter Moseley  wrote:
> 
> Is there a straight-forward way to estimate the amount of missing electron 
> density that a particular protein structure is missing based on the 
> difference between Fo and Fc?
> 
> It appears that the normalization of the Fc due to the employing of a maximum 
> entropy method that keeps Fo and Fc comparable to the standard deviation of 
> Fo would make this difficult. 
> Or am I missing something?
> 
> Cheers,
> Hunter
> 
> -- 
> Hunter Moseley, Ph.D. -- Univ. of Kentucky
> Associate Professor, Dept. of Molec. & Cell. Biochemistry / Markey Cancer 
> Center
> / Resource Center for Stable Isotope Resolved Metabolomics
>  Not just a scientist, but a fencer as well.
>My foil is sharp, but my mind sharper still.
> ---
> Email: hunter.mose...@uky.edu  (work)   
> hunter.mose...@gmail.com  (personal)   
> Phone: 859-218-2964 (office)   859-218-2965 (lab)   859-257-7715 (fax)
> Web: http://bioinformatics.cesb.uky.edu/ 
> Address: CC434 Roach Building, 800 Rose Street, Lexington, KY 40536-0093

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] Estimating the amount of missing electron density for a model

[Francis Reyes]

This is a good point, the difference between Fo and Fc can be great if Fc is 
actually missing (an 'incomplete' structure). And of course this wreaks havok 
in defining the maskf for bulk solvent, and refinement, etc. Incomplete can be 
missing whole parts of the protein (say in model building) or it could mean 
looking for density of a surface-bound ligand (the recent discussion of masking 
out the suspect region is implemented in BUSTER and more recently, phenix's 
POLDER maps). 

However, when it comes to the 'refinement' of incomplete structures, I've found 
that low resolution probability distributions for the missing atoms can improve 
the refinement and the resultant map via the  'MissingAtoms' channel of 
BUSTER-TNT during refinement[1]. There was also a phenix.refine 
use_statistical_model_for_missing_atoms from Tom Terwilliger, where the missing 
density is obtained from RESOLVE density modified maps. 

Of course, the best case scenario is to have an external source of phase 
information (say experimental phasing) and the incomplete Fc has less of an 
effect in the refinement and map quality. 


[1] Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2210-21. Epub 
2004 Nov 26. Refinement of severely incomplete structures with maximum 
likelihood in BUSTER-TNT. Blanc E, Roversi P, Vonrhein C, Flensburg C, Lea SM, 
Bricogne G.


Just my 0.02,


F

On Feb 21, 2017, at 4:53 PM, Hunter Moseley  wrote:

> Is there a straight-forward way to estimate the amount of missing electron 
> density that a particular protein structure is missing based on the 
> difference between Fo and Fc?
> 
> It appears that the normalization of the Fc due to the employing of a maximum 
> entropy method that keeps Fo and Fc comparable to the standard deviation of 
> Fo would make this difficult. 
> Or am I missing something?
> 
> Cheers,
> Hunter
> 
> -- 
> Hunter Moseley, Ph.D. -- Univ. of Kentucky
> Associate Professor, Dept. of Molec. & Cell. Biochemistry / Markey Cancer 
> Center
> / Resource Center for Stable Isotope Resolved Metabolomics
>  Not just a scientist, but a fencer as well.
>My foil is sharp, but my mind sharper still.
> ---
> Email: hunter.mose...@uky.edu (work)   hunter.mose...@gmail.com (personal)   
> Phone: 859-218-2964 (office)   859-218-2965 (lab)   859-257-7715 (fax)
> Web: http://bioinformatics.cesb.uky.edu/
> Address: CC434 Roach Building, 800 Rose Street, Lexington, KY 40536-0093



-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

--

[ccp4bb] PDRA position available in Leeds

[Glyn Hemsworth]

Dear All,

Please find details below for a PDRA position available at the University of 
Leeds. Please note the deadline for applications is 21st March 2017.

The full candidate brief can be downloaded from the following web address:

https://jobs.leeds.ac.uk/FBSMB1100

To apply for the position please register for an account on the Leeds website 
and submit a covering letter, together with a CV including details of at least 
two references.

Funded by the BBSRC the position is available for an initial 2 year period, 
with the possibility of extending to 4 years. The position will suit someone 
interested in redox proteins, structural biology and biotechnology. Please feel 
free to get in touch with informal enquiries about the role. Thanks and best 
wishes,

Glyn

–
Glyn Hemsworth
Astbury Building, Room 10.107
Faculty of Biological Sciences,
University of Leeds, Leeds, LS2 9JT, UK
Tel: (+44) 0113 3434 349
Email: g.r.hemswo...@leeds.ac.uk

Research Fellow

Are you an ambitious researcher looking for your next challenge? Do you have an 
established background in Structural Biology, Biochemistry or Chemistry? Do you 
want to further your career in one of the UKs leading research intensive 
Universities?

You will investigate the structure and function of a range of proteins, and 
their complexes, as potential activators of a class of enzymes known as lytic 
polysaccharide monooxygenases (LPMOs), (find out more in this recent 
review).
 LPMOs have emerged as key enzymes in biomass conversion, both in nature and in 
the applied setting of a biorefinery. Using a combination of structural, 
biophysical, and biochemical methods we are seeking to characterise the 
molecular mechanisms used in nature to shuttle electrons to LPMOs. The 
knowledge gained from this research is of considerable interest from both a 
fundamental and applied standpoint and will ultimately be used in protein 
engineering approaches to produce more efficient enzyme systems to be used in 
the biorefinery.

Funded by the BBSRC, you will be based in the newly established laboratory of 
Dr Glyn Hemsworth in the Faculty of Biological Sciences and the Astbury Centre 
for Structural Molecular Biology. With the recent £17 million investment in the 
Astbury Biostructure lab to complement the already superb infrastructure for 
structural molecular biology, Leeds represents the perfect environment for you 
to learn new techniques whilst contributing vital knowledge towards realising 
the development of lignocellulosing biofuels as a more sustainable energy 
source for the future.

You should have, or be close to completing a PhD in Structural Biology, 
Biochemistry, Chemistry or a related discipline, where practical experience of 
cryo-electron microscopy or X-ray crystallography is essential. You will be 
expected to show enthusiasm for engaging in inter-disciplinary research and 
expanding your knowledge and expertise into new areas. Experience of studying 
protein-protein interactions would also be an advantage.

To explore the post further or for any queries you may have, please contact:

Glyn Hemsworth

Tel: 0113 343 4349, email: 
g.r.hemswo...@leeds.ac.uk


Location:   Leeds - Main Campus
Faculty/Service:Faculty of Biological Sciences
School/Institute:   School of Molecular & Cellular Biology
Category:   Research
Grade:  Grade 7
Salary: £32,004 to £38,183 p.a.
Contract Type:  Fixed Term (Fixed term for 2 years due to funding - optional 4 
years)
Closing Date:   Tuesday 21 March 2017
Reference:  FBSMB1100

Re: [ccp4bb] Dimer in SDS-PAGE

[Bert Van-Den-Berg]

like others I'm not clear why you care where your protein runs on SDS-PAGE. I 
think the band you're seeing is in fact the tetramer, suggesting your protein 
(like KcsA) is very stable. Helical membrane proteins often migrate faster than 
expected (by their Mw) on SDS-PAGE.

Also, never boil helical membrane protein samples, they will aggregate.


bert



From: CCP4 bulletin board  on behalf of amit gaur 

Sent: 21 February 2017 22:22
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Dimer in SDS-PAGE

Hi all,
  I am trying to purify a potassium ion channel from insect cell using 
baculovirus expression system. I am not seeing monomer of this protein in SDS 
instead a dimer appears.So,I increased DTT in SDS buffer but no change and 
dimer was intact. In size exclusion this protein appeared as a tetramer which 
is common oligomerizaton of potassium channel family with GYG motif. Can any 
body suggest what should I do in this case?

Thanks and regards,


--
Dr. Amit Gaur
Post Doctoral Researcher
PI: Dr. Ji-Fang Zhang
Thomas Jefferson University
1020, Locust Street, Suite 418
Philadelphia, PA 19107