Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Thomas Edwards]

Hi Rafael, Dom et al,

Indeed nickel eluted with imidazole stays on all your proteins with a His tag, 
and addition of DTT will make almost all proteins go brown and crap out at this 
point. But, as Dom says, addition of EDTA before the DTT will solve the 
problem. Most of the time…

If your protein goes brown after a nickel column and you thought it was 
something special to your protein, try again..!

If your protein can’t handle EDTA, try the pH trick suggested in another post.

If none of that works, then move to GST or some such…

Ed
https://www.ularkin.org/team-member/thomas-edwards/
Sent from my iPhone. On the run…

On 31 Oct 2023, at 16:48, Dom Bellini  wrote:

 Hi Rafael,

Once I inherited a protocol with a problem similar to yours and they told me 
that the precipitation was caused by nickel leaking out during the elution with 
250 mM imidazole. I am not sure whether this was true, however, their fix was 
to place something like 20 ul of 200 mM EDTA at the bottom of the collection 
tubes into which the protein was eluting. This indeed kept the protein soluble, 
at least long enough to dialyse or gel filtration.

Good luck!

D

On 31 Oct 2023, at 19:21, Rafael Marques  wrote:


CAUTION: This email originated from outside of the LMB.
Do not click links or open attachments unless you recognize the sender and know 
the content is safe.
.-owner-ccp...@jiscmail.ac.uk-.

Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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[ccp4bb] FW: Postdoc position in Structure-Based Cancer Drug Discovery - Astbury Centre, Leeds University

[Thomas Edwards]

 PLEASE DO NOT REPLY TO ME...

Please direct enquiries to Prof Alex Breeze:

E: a.l.bre...@leeds.ac.uk

T: +44 113 343 0087




Dear Colleagues,

***Postdoctoral Research Fellow in Structure-Based Cancer Drug Discovery***

There is an immediate opening in my lab at the Astbury Centre (University of 
Leeds, UK) for a versatile structural biologist to work on cancer drug 
discovery in a collaboration with the Northern Institute for Cancer Research at 
Newcastle University.

The position, which is funded for 2 years by Cancer Research UK (CRUK), is for 
development of small molecule inhibitors of the Ras activating protein, SOS. 
You will preferably be an experienced X-ray crystallographer, with 
complementary skills and expertise in other structural and biophysical methods, 
including NMR, SPR or ITC. The project will also involve implementation of 
cell-based assays in conjunction with the lab of Dr Darren Tomlinson (Leeds). 
Medicinal chemistry expertise and resource will be provided by our Newcastle 
collaborators (Prof Mike Waring).

The successful applicant will have access to the Astbury Centre’s excellent 
structural biology facilities including a state-of-the-art crystallisation 
suite and cryoprobe-equipped 950 MHz NMR.

For more details and to apply please follow this link:
https://jobs.leeds.ac.uk/vacancy.aspx?ref=FBSMB1168

…or for informal enquiries please contact me directly.

Best,
Alex

--
Prof Alexander L Breeze
Professor of Biomolecular NMR
Director of NMR, Astbury BioStructure Laboratory
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
University of Leeds
Leeds
LS2 9JT, UK
E: a.l.bre...@leeds.ac.uk
T: +44 113 343 0087




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Re: [ccp4bb] [OT] Structure-related pun needed urgently

[Thomas Edwards]

“Can I speed up structure solution using different phases”…??

I’ll get my coat.

[Gerard - what is the audience? Crystallographers vs general public might make 
a difference..!]


Ed

T.A.Edwards Ph.D.
Associate Professor of Biochemistry
Deputy Head of School
_
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
===
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
Faculty Staff 
Profile
Astbury Centre Web 
Page
[signature_1707952708]
Perturbation of Protein-Protein Interactions

Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka

[/Users/bmbtae/bmbtae/0_Docs/Astbury/0_AstburyConversation/2020/01784_Astbury 
Conversation 2020 email signature 
(WR).png]

From: CCP4 bulletin board  on behalf of "Daniel M. 
Himmel, Ph. D." 
Reply-To: "Daniel M. Himmel, Ph. D." 
Date: Thursday, 15 August 2019 at 15:57
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] [OT] Structure-related pun needed urgently

You're welcome to boo this one down, but how about something like, "With a some 
coaxing,
a protein can be X-cited to expose its structure."  I think that's poor, but 
maybe it's a starting point.

-Daniel


On Thu, Aug 15, 2019 at 7:42 AM Gerard DVD Kleywegt 
mailto:ger...@xray.bmc.uu.se>> wrote:
Dear CCP4BB-ers,

Once again I turn to you in my hour of need. I *urgently* need a
side-splittingly funny, and ideally punny, structure-related
sentence/statement/claim/expression to put in a speech bubble attached to a
life-size bobblehead version of yours truly (don't ask)!

I know there are some very funny people on CCP4BB. The best I've been able to
come up with myself so far is: "Protein structures are beautiful, but I try to
keep it platomic" - which is pretty lame, I know.

Many thanks in advance!

--Gerard

**
Gerard J. Kleywegt

   http://xray.bmc.uu.se/gerard   
mailto:ger...@xray.bmc.uu.se
**
The opinions in this message are fictional.  Any similarity
to actual opinions, living or dead, is purely coincidental.
**
Little known gastromathematical curiosity: let "z" be the
radius and "a" the thickness of a pizza. Then the volume
 of that pizza is equal to pi*z*z*a !
**



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[ccp4bb] Rigaku system up for grabs

[Thomas Edwards]

Dear CCP4bb

We are going through a large lab refurbishment programme and that, along with 
space costings for facilities, mean that sadly we will not be able to keep our 
local generator & detector.

If anybody can come to Leeds and take it all away (free!) then please do so!

The system:
Rigaku MicroMax 007 High-Flux generator with Raxis IV++ detector
About 6 years old.

Please do get in touch.

Ed

T.A.Edwards Ph.D.
Associate Professor of Biochemistry
Deputy Head of School
_
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
===
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
Faculty Staff 
Profile
Astbury Centre Web 
Page
[signature_1395366918]
Perturbation of Protein-Protein Interactions

Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka

[/Users/bmbtae/bmbtae/0_Docs/Astbury/0_AstburyConversation/2020/01784_Astbury 
Conversation 2020 email signature 
(WR).png]



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Re: [ccp4bb] OFF TOPIC

[Thomas Edwards]

You don’t say so, but one assumes that you have a BAP tag on the protein, and 
co-express a biotin ligase such as BirA?


Ed

T.A.Edwards Ph.D.
Associate Professor of Biochemistry
Deputy Head of School
_
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
===
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
Faculty Staff 
Profile
Astbury Centre Web 
Page

Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[signature_1229172001]
Perturbation of Protein-Protein Interactions


From: CCP4 bulletin board  on behalf of Anamika Singh 

Reply-To: Anamika Singh 
Date: Tuesday, 13 November 2018 at 10:15
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] OFF TOPIC

Hi All,

I am purifying the biotinylated protein (cloned into the pET28a vector) using 
Avidin beads. Since I need the protein for SPR but when I used the purified 
protein to interact with Streptavidin coated onto the SPR chip. There was no 
signal. Can anybody tell me why is it so or how can I make sure that the 
purified protein is biotinylated enough to interact and give the signal? 
Because when I ran the SDS-PAGE there was a band of purified protein.

I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM 
Biotin).
PS: I have included the biotin during overexpression of the protein also.

Please suggest.

Thanks
--
Anamika
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel




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[ccp4bb] Asbury Conversation April 16-17th. Abstract deadline approaching

[Thomas Edwards]

Dear Structural Molecular Biologists,


There are still some places available to register for the Astbury Conversation, 
so if you or any colleagues who would benefit from coming – full details are 
available on the web site http://www.astburyconversation.leeds.ac.uk/

With just over eight weeks to go until the two-day Conversation, I wanted to 
remind you of the approaching deadline for talk/poster abstract submissions.

Abstracts and posters
If you have not yet registered or submitted an abstract, please make sure you 
do so no later than the deadline of Wednesday 28 February.

Symposium talks will be selected from submitted abstracts, and those speakers 
who have been chosen to give a talk, will be notified by Friday 23 March.


Financial support available
We are delighted to confirm that two of our partners have agreed to provide 
bursaries, to help towards the costs of travel and accommodation. The British 
Biophysical Society is providing two bursaries of £200 each for UK registrants 
and Astrazeneca two bursaries of £250 for international registrants.

To apply to be considered for one of these, please email astb...@leeds.ac.uk 
with details of your abstract, a short summary of your reasons for applying for 
support and specify which bursary you are applying for.





Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, Director of Research, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
PoPPI (Perturbations of Protein-Protein Interactions) project: 
http://www.poppi.website/
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[id:image001.png@01D336AA.C1A20F40]

Re: [ccp4bb] "Atomic resolution"

[Thomas Edwards]

Dear Jacob,

Ah... this old chestnut!

Current EM people say that they are at atomic resolution because they are 
building atomic models (naive??).

I have been criticised in the past for using the term with say 2.2A diffraction 
data. By co-authors and reviewers alike. When I was young and naive.

My (current) definition would be yours - visible with data.
I think 1.5A is about right for X-ray. Maybe higher res?

I’m sure there are lots of rigorous ways to think. I probably haven’t taken 
that route. However, I think it is a semantic problem that might benefit from 
some disambiguation rather than rigour.

It depends why you are asking the question...

Sorry..!

Ed is: Out and about...
Sent from iPhone6sPlus.

On 11 Jan 2018, at 19:31, Keller, Jacob 
> wrote:

Dear Crystallographers,

Has there been a consensus as to what is meant by “atomic resolution?” Seems 
like the term is taken by various practitioners to mean different things.

A related question: at what resolution are atoms “visible” using only the data? 
I have an empirical feeling that this would be around 1.5 Ang Bragg spacings, 
but on the other hand, one can contour up most maps and see individual atom 
peaks. I would be interested to hear a more rigorous way to think about this.

All the best,

Jacob Keller

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
(571)209-4000 x3159
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

[ccp4bb] structural biologist to work on cancer drug discovery

[Thomas Edwards]

Please respond direct to Alex.


Dear Colleagues,

I have a 16 month postdoctoral position available immediately in my lab at the 
Astbury Centre, University of Leeds, for a versatile structural biologist to 
work on cancer drug discovery in a collaboration with the Northern Institute 
for Cancer Research at Newcastle University. For more details and to apply 
please follow this link:

http://jobs.leeds.ac.uk/FBSMB1123

…or for informal enquiries please contact me directly.

Best,
Alex

--
Prof Alexander L Breeze
Professor of Biomolecular NMR
Director of NMR, Astbury BioStructure Laboratory
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
University of Leeds
Leeds
LS2 9JT, UK
E: a.l.bre...@leeds.ac.uk
T: +44 113 343 0087



NB click on the link below to register for the Astbury Conversation. Abstract 
submissions now open.


T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, Director of Research, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
PoPPI (Perturbations of Protein-Protein Interactions) project: 
http://www.poppi.website/
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D36F3A.9F1339E0]

[ccp4bb] registration for the Astbury Conversation 2018 is now open

[Thomas Edwards]

Dear CCP4 members,

Following the huge success of the inaugural Astbury Conversation 2016, we are 
delighted to announce that registration for the Astbury Conversation 2018 is 
now open!!

The event will bring together some of the world’s most prominent 
molecular/chemical/structural biologists for a two day symposium and public 
event, with our keynote speaker being Stanford University’s Nobel Laureate 
Professor Brian Kobilka.

Other confirmed speakers include:
Professor David Agard (University of California)
Professor Dame Carol Robinson (University of Oxford)
Professor Hagan Bayley (University of Oxford)
Professor Katrin Rittinger (The Francis Crick Institute, London)
Professor John Briggs (University of Cambridge)
Professor Riki Eggert (King’s College, London)
Professor Babis Kalodimos (University of Minnesota)
Professor Jim Naismith (University of Oxford and Director of the Research 
Complex at Harwell)
Professor Matthias Rief (Technical University of Munich)

The registration fee is just £49 (no accommodation) or £139 (including 
overnight accommodation). This includes access to all talks, event materials 
and meals, including beer and pizza at the poster evening.

Places are limited, and the event is now being advertised both nationally and 
internationally- so please book your place ASAP at 
http://www.astburyconversation.leeds.ac.uk/index.php


We had overwhelmingly positive feedback from the last event – and the following 
is just one example:
“Just a brief message to thank you and say how much I enjoyed the Astbury 
Conversation. It was one of the best meetings I've been to in recent years - a 
great set of speakers (some in areas I know well and others who widened my 
horizons), a very positive environment and a length of meeting that fits in 
well with busy teaching schedules.”
Delegate from 2016 Astbury Conversation


Would you like to know more? You can find more details at 
www.astburyconversation.leeds.ac.uk
 and if you have any questions, please email Lucy Gray at 
l.v.g...@leeds.ac.uk.

We look forward to welcoming you to the 2018 Astbury Conversation.

Best wishes,


Sheena & Ed

Sheena E. Radford, FMedSci, FRS
Director, Astbury Centre for Structural Molecular Biology,
University of Leeds

Ed
Symposium Convenor, Astbury Conversation 2018

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, Director of Research, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
PoPPI (Perturbations of Protein-Protein Interactions) project: 
http://www.poppi.website/
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D33C68.303A5170]

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Thomas Edwards]

A few tips, some/all of which may be relevant. Or not…

If you are in 96 or 384 well plates, they vary. We tried quite a few batches 
and brands before settling on one.

Keep your salt concentration as low as possible.

You should be able to measure Kd in a range of about 1nM to low uM. Outside 
that range is harder, or possibly impossible.

Buffers matter.
RNA binding proteins have preferred phosphates, PPIs Tris.
Some triton and/or some BSA can help.

All proteins are different, and each likes some or all or none of the above…..

Ideally you should convert polarization to anisotropy. Simple enough – but some 
referees can get picky…

Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D30233.9026B5C0]

From: CCP4 bulletin board  on behalf of Mohammad Khan 

Reply-To: Mohammad Khan 
Date: Friday, 21 July 2017 at 14:32
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all,

I am trying to measure the difference in polarization upon the binding of the 
DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions 
of my protein to it (100 microM to 1 nM). I do get a decrease in difference of 
polarization with decrease in protein concentration. However, the results are 
difficult to reproduce and also vary greatly within triplicates of an 
experiment.

Similar observations have been observed by my colleagues with their proteins.

Are there any tips or precautions to keep in mind while setting up these 
reactions?

Looking forward for suggestions.

Thank you.

[ccp4bb] 15 positions for Early Stage Researchers (ESR) in virology

[Thomas Edwards]

As part of the “HONOURS” ITN EU network, there are 15 early stage researcher 
(studying for a PhD) positions available in Belgium, Germany, the Netherlands, 
Spain, Switzerland and the United Kingdom.
See details below, and specific details of those hosted at the University of 
Leeds.

Ed




Early Stage Researchers in HONOURs
HONOURs is an Innovative Training Network (No: 721367) on host switching 
pathogens, infectious outbreaks and zoonosis: https://www.honours.eu/home. 
There are 15 positions for Early Stage Researchers (ESR) at the interface of 
veterinary and human health, virology, biostatistics, and pathogen discovery, 
and we are looking for highly motivated and talented students with a MSc degree 
in Life Sciences who are interested in viruses and infectious diseases. We 
offer challenging research projects to lead to a dissertation (PhD thesis) at 
high profile universities, research institutions and companies, located in 
Belgium, Germany, the Netherlands, Spain, Switzerland and the United Kingdom. 
The framework of the network, the research projects, and the application form 
to apply for the ESR positions are presented at the HONOURs website: 
https://www.honours.eu/vacancies/general-information. Recruitment has started 
on 15th April 2017.


[CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 80]


ESR11: Viral nucleocapsid proteins, their structure and novel diagnostic assays
Host Institute: The University of Leeds, Leeds, United Kingdom.
The University of Leeds (UL) is a member of the Russell Group of 
Research-intensive Universities and has a student population of over 33,000 of 
which around 9,000 are postgraduates. UL is home to the Astbury Centre for 
Structural and Molecular Biology (ACSMB), which comprises over 70 principal 
investigators and 350 researchers with expertise in biochemistry, structural 
biology and molecular cell biology. ACSMB is one of the largest centres for 
virus research within the UK, having 13 virus-themed group leaders and over 70 
postdoctoral and postgraduate researchers studying viruses. Of significance to 
this project is the recent purchase by UL of two Titan Krios microscopes with 
Falcon III detectors, a £10m investment by the university in ASCMB facilities. 
ASCMB and UL offers an exceptional research environment for talented 
individuals that are looking for a place where they can excel in virology and 
structural biology.
Project description: Detailed characterisation of an emerging pathogen is a 
critical step in mitigating potential disease outcomes, and we believe one way 
to achieve this is a better understanding of the structure and function of 
viral components. Dr J. N. Barr has over 20 years research experience of RNA 
viruses, and recent work together with Dr T. A. Edwards has focussed on 
understanding the structure/function relationship of many viral proteins. This 
work has involved structural analysis by X-ray crystallography and electron 
microscopy, and functional analysis using both biochemical and biophysical 
means, as well as reverse genetics with the rescue of replicons and infectious 
viruses. This ESR project will focus on characterising a critical component of 
all negative stranded RNA viruses known as the ribonucleoprotein (RNP), which 
comprises the viral RNA genome in complex with multiple viral proteins. The 
viruses we will study include multiple zoonotic negative stranded RNA viruses 
from the Bunyaviridae, Arenaviridae and Paramyxoviride families. The ESR will 
use both cryo-EM and crystallography to solve the high resolution structures of 
RNPs generated from reconstituted components and also extracted from infectious 
viruses. In addition, the ESR will also have the opportunity to extend their 
work to include RNPs and other proteins from zoonotic viruses that may emerge 
within the time frame of the HONOURs project. The ESR appointed to this 
position (ESR-11) will also work closely with another ESR at UL (ESR-14), who 
will develop novel diagnostic assays and there will be extensive exchange of 
antigen and antibody reagents between the ESRs to build synergy. The ESR-11 
will join both the Barr and Edwards laboratories, that together comprise a 
skilled team of 12 PhD students, one technician and three post doctoral fellows.
HONOURs is looking for a highly motivated and enthusiastic researcher with a 
flexible, pro-active team spirit and a recent MSc degree (within the first four 
years after graduation) in the field of biochemistry, molecular biology, 
virology or life sciences. Interest and experience in infectious diseases and 
structural biology is an advantage. Proficiency in English speaking, reading, 
and writing is a requirement. The applicant should not have obtained her/his 
degree in the United Kingdom (mobility rule EU). Read the further requirements 
at https://www.honours.eu/vacancies/general-information
Supervisors: Drs John N Barr and Thomas A Edwards; Duration: 36 months Doctoral 
defence at the 

[ccp4bb] Research Fellow in Chemical Biology, Leeds

[Thomas Edwards]

Dear CCP4 members,

A reminder of the 30th March closing date for a research fellow position in my 
lab to collaborate with chemists here at Leeds to design inhibitors of 
protein-protein interactions:
http://jobs.leeds.ac.uk/FBSAS1006

Closing Date:

Thursday 30 March 2017



We are looking for an enthusiastic structural biologist with interests in 
synthetic and medicinal chemistry.

Please Email with informal enquiries. Formal applications must be made through 
the web site.

Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D2A6E9.F0271690]

[ccp4bb] Research Fellow in Structural/Chemical Biology

[Thomas Edwards]

Details below of a position now open in my lab as part of a project to design 
and test inhibitors of protein-protein interactions.
Please Email me with any informal enquiries.



Research Fellow in Structural Molecular Biology

Are you looking to apply your skills in Structural Biology to the development 
of new approaches for discovery of protein-protein interaction inhibitors?

Protein-protein interactions (PPIs) control all cellular processes relevant to 
health and disease. Selective inhibition of individual PPIs would thus 
facilitate both a greater understanding of biological mechanisms and provide 
new opportunities for therapeutic intervention. Presently, PPI inhibitors 
represent a minute fraction of current small molecule drugs, this is largely 
because of specific challenges associated with development of inhibitors for 
these targets.
The Perturbation of Protein-Protein Interactions 
(PoPPI) project is a major £3.4 million 
five-year collaborative research programme led by Professor Andy 
Wilson, funded by 
the Engineering and Physical Sciences Research Council 
(EPSRC), and bringing together the University of 
Leeds, the University of 
Bristol, the Northern Institute of Cancer 
Research (Newcastle University) and drug discovery 
organisations, AstraZeneca and 
Domainex. This large and diverse programme focuses 
on developing, validating and exploiting new tools to discover inhibitors of 
PPI’s. This position will use structural biology and biophysics to assess the 
application of our methods for preparing inhibitors of multiple PPIs. The 
position will provide multiple opportunities for personal development, as well 
as the possibility of research secondments at Bristol and our drug discovery 
partners.
You should have, or be about to obtain, a PhD in Biochemistry or related 
biological science with expertise in Structural Chemistry. You will work 
closely, interactively and collaboratively with project team in an 
interdisciplinary setting, so you will need excellent communication skills, the 
ability to work under pressure and to meet deadlines.


To view the advert with details of how to apply use the link below:

http://jobs.leeds.ac.uk/FBSAS1006


Closing Date:

Thursday 30 March 2017



Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D2940F.1D134C70]

Re: [ccp4bb] metal ion coordination

[Thomas Edwards]

Hi David and Faisal,

That’s a great page. I would also like to recommend the “CheckMyMetal” server:

http://csgid.org/csgid/metal_sites/

In addition to the Harding reference one of my favorites is this one:

Zheng,H. et al. (2008) J. Inorg. Biochem., 102, 1765-1776.
Data mining of metal ion environments present in protein structures. 
http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/18614239

Tom

Thomas Edwards, Ph.D.
Director, Technology  Client Solutions
Co-PI, Seattle Structural Genomics Center for Infectious Disease (SSGCID)
Head, Infectious Disease Practice Area
Emerald Bio
Office: (206) 780-8949
Cell:(206) 495-1300
tedwa...@embios.commailto:tedwa...@embios.com

Boston area: 3 Preston Court, Bedford, MA 01730 USA
Seattle area: 7869 NE Day Road West, Bainbridge Island, WA 98110 USA

www.embios.comhttp://www.embios.com/
(855) 4EMBIOS (436-2467)




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David 
Briggs
Sent: Thursday, April 17, 2014 1:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] metal ion coordination


Hi Faisal,

Take a look at Marjorie Harding's website

http://tanna.bch.ed.ac.uk

Loads of information there.

Hth,

Dave

Dr David C Briggs PhD
http://about.me/david_briggs
On 17 Apr 2014 21:16, Faisal Tarique 
faisaltari...@gmail.commailto:faisaltari...@gmail.com wrote:
Dear all

Can anybody please explain what is the classical metal ion coordination for 
Mg2+, Ca+ and Na+ with Oxygen atom and the average distance with these metal 
ions..does the distance vary with the type of metal ion and its coordination 
with oxygen atom..what is the best way to identify the correct metal ion in the 
electron density in the vicinity of negatively charged molecule mostly oxygen 
containing molecule..In one of my paper the reviewer has asked me to check 
whether the octahedrally coordinated Mg+ is  Ca+ ion..and similarly raised 
doubt about the identity of the Na+ ion as well..his argument was based on 
metal ion to oxygen distance..I am attaching the figure with this mail..i 
request you to please shed some light on this area and help me in clearing some 
doubts regarding this.

--
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] Lecture in X-ray crystallography, University of Leeds

[Thomas Edwards]

Please see the link below for an advert for a new Lectureship in Macromolecular 
X-Ray Crystallography at the University of Leeds.

http://jobs.leeds.ac.uk/fe/tpl_universityofleeds01.asp?s=4A515F4E5A565B1Ajobid=110947,6951214874key=11037106c=612123217223pagestamp=sepwjldjailxhilecl

Job Summary


You will be appointed in the School of Molecular  Cellular Biology, where you 
will join a dynamic School of around 40 academic staff with research interests 
that span Structural Biology, Molecular Biology, Cell Biology, Virology, 
Bacteriology and Computational Biology. The School has excellent research 
facilities including a recently upgraded X-ray facility, along with newly 
updated facilities for NMR, electron microscopy, mass spectrometry and high 
performance computing.

You should have a PhD in a relevant area as well as a successful research track 
record in X-ray crystallography applied to important questions in the 
biological sciences. We are particularly keen to attract candidates who 
complement our recent strategic investments in chemical biology, inhibitor 
discovery and macromolecular recognition, but candidates with interests in any 
area of structural molecular biology that fit within the School’s research 
strengths are welcome to apply.

University Grade 8 (£37,756 - £45,053 p.a.)

Informal enquiries may be made to Professor David Westhead, tel +44 (0)113 343 
3116, email d.r.westh...@leeds.ac.ukmailto:d.r.westh...@leeds.ac.uk

Closing Date: 7 April 2014


Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae
--Science is always wrong. It never solves a problem without creating ten more. 
~George Bernard Shaw

[ccp4bb] Bper lysis reagent recipe

[Thomas Edwards]

Lovely CCP4BB Members,

Hopefully not too far off topic:

It appears that our sonicator is, ironically, bust.

Whilst I wait for a new one, and/or some B-PER reagent, does anybody have a 
home made detergent lysis reagent (something like B-PER) recipe?
Only E. coli to bust open (for now).

Many Thanks
Ed


T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae
-- There is something fascinating about science.  One gets such wholesale 
returns of conjecture out of such a trifling investment of fact.  ~Mark Twain

[ccp4bb] post-doctoral opportunity at the University of Leeds

[Thomas Edwards]

Dear CCP4bb,

If you are a talented and motivated postdoctoral structural biologist, or soon 
to be, or know somebody who is…


Project Title: Structure and function of the essential M2-1 protein of human 
respiratory syncytial virus

Our laboratories are interested in understanding the structural and cellular 
biology of a variety of negative strand RNA viruses. One of these viruses is 
human respiratory syncytial virus (HRSV), which is the leading cause of lower 
respiratory tract illness in young children and the immunocompromised, and has 
been linked to adult asthma. We recently determined the first X-ray crystal 
structure of the HRSV M2-1 protein, which is an essential transcriptional 
anti-terminator (Tanner et al., PNAS, 2014). The current project seeks to 
further understand the essential role of M2-1 in viral mRNA transcription, and 
will involve both X-ray crystallography and cell biological approaches using 
live recombinant virus and genome analog analysis.

You will have a PhD (or be close to completion) in Structural Molecular Biology 
or a closely allied discipline, together with a proven track record of protein 
structure solution using X-ray crystallography. Experience of working with 
viruses in mammalian cell culture is highly desirable.

University Grade 7 (£30,728 - £36,661 p.a.) Due to funding limitations an 
appointment will not be made above £30,728 p.a.

Informal enquiries may be made to either Dr John N. Barr, email 
j.n.b...@leeds.ac.ukmailto:j.n.b...@leeds.ac.uk or Dr Thomas Edwards, email 
t.a.edwa...@leeds.ac.ukmailto:t.a.edwa...@leeds.ac.uk.

Closing Date: 13 February 2014

http://jobs.leeds.ac.uk/fe/tpl_universityofleeds01.asp?s=4A515F4E5A565B1Ajobid=108922,2351796198key=9056137c=344787029814pagestamp=sedylihlceulaesvjp


Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae
Live as if you were to die tomorrow. Learn as if you were to live forever. 
Mahatma Gandhi

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Thomas Edwards]

Hi Shane,

One of my favorite examples of large structural differences between molecules 
in the asymmetric unit have is the crystal structure of the L1 ribozyme (PDB ID 
2oiu). The Q and P chains have two stems which crudely co-axially stack and a 
third stem which appears 180 degrees in the opposite direction between the two 
molecules in the asymmetric unit. See Figure 2C of the paper: Robertson, M.P.  
Scott, W.G. The structural basis of ribozyme-catalyzed RNA assembly. Science 
2007, 315, 1549-1553; PubMed ID 17363667. Only one conformation appears to be 
active.

Thomas Edwards

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Aaron 
Thompson
Sent: Monday, January 27, 2014 5:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Examples of multiple ASU copies with different 
conformations

The structure of kappa opioid receptor fused with T4 lysozyme (4DJH) contains 
two copies in the ASU - each copy displays a different orientation between the 
receptor and lysozyme.

On Mon, Jan 27, 2014 at 10:08 AM, Shane Caldwell 
shane.caldwel...@gmail.commailto:shane.caldwel...@gmail.com wrote:
Hi ccp4bb,
I'm putting together a talk for some peers that highlights strengths and 
weaknesses of structural models for the outsider. For one point, I'd like to 
find some examples of proteins that show very different conformations between 
different copies in the ASU. One example I know of is c-Abl (1OPL), which 
crystallizes with both autoinhibited and active forms in the ASU, with 
dramatically different domain organization. I'd like to find some additional 
examples - can anyone suggest some other structures that have multiple copies 
with large structural variations?
Thanks in advance!

Shane Caldwell
McGill University

[ccp4bb] PhD studentship opportunities in Leeds

[Thomas Edwards]

Dear BB,

If you are, or know, a keen, talented, motivated student looking for a PhD, 
then there are several opportunities within the Faculty of Biological Sciences 
at the University of Leeds.
One project involves the use of stapled peptides to investigate inhibition of 
protein-protein interactions in cancer development and progression, another 
will look at Structure based drug design of anti vitals against a Hemorrhagic 
Fever Virus.
See:
http://www.fbs.leeds.ac.uk/staff/tae/EPSRC_Advert_JB_TE_RF.pdf
http://www.fbs.leeds.ac.uk/staff/tae/EPSRC_Advert_TAE_AJW.pdf
For more info. The deadline is short (20th Feb), appointments will be 
competitive,and the student must be in place at Leeds by April…

Email me with informal enquiries.

Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae
--You can't possibly be a scientist if you mind people thinking that you're a 
fool. - Wonko the Sane

[ccp4bb] PPI-Net Young Researchers Meeting

[Thomas Edwards]

Dear CCP4bb,

A symposium on protein-protein interactions organised by young researchers from 
around the UK.
Will probably appeal to some from the UK, but obviously all welcome.
Please respond to the organising committee (yr2...@ppi-net.org) rather than to 
me.

Thanks
Ed




 This symposium aims to provide a platform for early career Protein-Protein 
Interaction researchers from a broad range of backgrounds to come together, 
share their work and discuss the latest research. In addition to our excellent 
guest speakers there will also be opportunities for young researchers to 
present their work through short talks and posters chosen from abstracts. 
Please submit your abstract when you register for the symposium.

Plenary Lecture: Paramjit Arora (NYU)

Invited speakers: Dan Davis (Imperial college); Chun-wa Chung (GSK).


Please register at ppi-net.org/events.

If you have any questions please contact us at yr2...@ppi-net.org


T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Lecturer in Biochemistry
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
-- The universe is full of magical things patiently waiting for our wits to 
grow sharper.  ~Eden Phillpotts

[ccp4bb] protein stain, B-PER

[Thomas Edwards]

Dear BB,

Apologies for being mildly off topic.
Maybe.


 1.  We are trying to express (in E. coli) a protein which appears to be quite 
sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER 
Bacterial Protein Extraction Reagents are designed to extract soluble protein 
from bacterial cells without harsh chemicals or mechanical procedures like 
sonication), but would like to try a variety of similar things if possible. 
Any advice from the community out there? Anybody know what goes into B-PER or 
similar things (I know there's some Dnase and lysosyme in there – but which 
detergents are compatible with Ni, GST, how much do you need etc)??
 2.  Staining SDS gels. There are various concerns from lab members about 
safety re methanol in stains, microwaving stains etc etc. Instant Blue claims 
to have none of these problems.  Quote: Protein gel staining takes around 15 
minutes without the need to wash, fix, microwave or destain. But again, I'd 
like to try things to see if they work for us (before spending cash - yes, I am 
spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, 
non-microwave, non–destain protein gel stains? Have tried home made colloidal 
coomassie but our protocol still requires fixes and washes that made it not 
really worth while.

Happy to collate thoughts on replies offline and post summary.

Many thanks
Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Lecturer in Biochemistry
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
-- No one should approach the temple of science with the soul of a money 
changer.  ~Thomas Browne

[ccp4bb] PhD studentship in Structural Molecular Biology at Leeds Oct 2011

[Thomas Edwards]

Dear BB,

Please forward to any potential PhD applicants.

Thanks
Ed



PhD studentship in Structural Molecular Biology in collaboration with 
AstraZeneca.
Part of an integrated approach to discovering cell-permeable inhibitors of 
alpha helix-mediated protein-protein
interactions.

See
http://www.bmb.leeds.ac.uk/staff/tae/AZ_Studentship_inStrucBiol_Ad.pdf
For details.


Supervisory team:
Dr Thomas Edwards, Dr Andy Wilson, Professor Adam Nelson and Dr Stuart Warriner.

Protein−protein interactions (PPIs) mediate many biological mechanisms,
and, potentially, represent exciting targets for the treatment of disease.
Although small molecule inhibitors of a few PPIs have been discovered,
the principles that underpin the discovery of such ligands, within the
boundaries of drug-like chemical space, are much less well established
than for conventional medicinal chemistry targets such as enzymes and
receptors. In addition, it is unlikely, for example, that current small
molecule collections are honed for screening against PPIs.
We will target three contrasting α-helix mediated PPIs of strategic interest
to AZ. The proposed programme will integrate the combined expertise of
the team at Leeds (and our collaborators at AZ) in the synthesis of both
helix mimetics, and small molecules of unprecedented scaffold diversity,
in combination with cutting edge structural biology.

The studentship will start by October 1st 2011 and the stipend
would be at the standard research council rate (~ £13,895 pa
for 2011/12). Applications, for this 3.5 year studentship which
will be accepted until the studentship is filled, should be
directed to Anna Luty, School of Chemistry, University of Leeds, Leeds LS2 9JT 
and can be made
online via the following link: 
http://www.chem.leeds.ac.uk/postgraduate-research/how-to-apply.html

Please contact Dr Thomas Edwards (t.a.edwa...@leeds.ac.uk) or Dr. Andy Wilson
(a.j.wil...@leeds.ac.uk) for further details about this project.

UK Research Council eligibility rules apply.


__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
-- There is something fascinating about science.  One gets such wholesale 
returns of conjecture out of such a trifling investment of fact.  ~Mark Twain

[ccp4bb] Free R with doubled cell edge

[Thomas Edwards]

Dear BB,

Thanks for all of your helpful and useful comments

Whilst there was some agreement that simulated annealing should be enough to 
solve any problems, we have taken perhaps the cautious approach.
We have produced a freeR set in the bigger unit cell using reindex as suggested 
by Eleanor:


This is easy to do
Reindex 2h,k,l then the cell will double; the FreeR will stay with the
reflection, and you can use those FreeRs to append to your new data in
the scala/truncate GUI.
All the unset ones (2h+1,k,l) will be given new FreeRs and the old ones
transferred.
Eleanor

Mostly for the reasons as outlines by Ethan:


One problem with shaking things up as you describe is that if
the original model was refined against higher resolution data
than your new data set, you will probably never get back to
the same model quality as you started with (see the ongoing
discussion in another thread).

And if the new data set is higher resolution, then you face the
same problem in reverse. If you want to take your eventual new,
higher resolution model back into the old cell you want
subsequent refinement to have unbiased Rfree on that end also.

Ethan


as we now have higher resolution data (than we had for the small unit cell 
before) for both the small and doubled unit cell - the highest resolution data 
is for the doubled unit cell with 4 chains, but we would like to refine in the 
smaller cell as well at some point for completeness.

As pointed out, there is a straight translation in the larger unit cell 
(doubled along a), h(odd) 0 0  reflections are weak, and Phenix Xtriage 
identifies the translation readily.
However, Phaser has correctly positioned 4 chains and we are happily refining 
in Refmac, so I think that should not be a problem.

Many thanks to all who commented on the BB and off line.

Cheers
Ed



__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
People who know nothing about cheeses reel away from Camembert, Roquefort, and 
Stilton because the plebeian proboscis is not equipped to differentiate between 
the sordid and the sublime. Harvey Day (1971)

 Dear BB Sages,

 I have a problem where I think I could very easily do the wrong thing.
 And I don't really want to do that...

 We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU 
 - Shelx, RESOLVE, ARPwARP. Cool.).
 In p21 30 109 65 90 105 90 at 2.5A

 However, we have now collected 1.9A data.
 In p21...
 60 109 65 90 107 90

 4 chains per AU instead of 2 with a doubling of a.

 Self rotations with the new data suggest 2 two-folds, one quite near 
 crystallographic.
 It seems that the doubling of the a edge is adding an NCS two-fold that is 
 almost crystallographic.

 Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would 
 like to use that model to do MR against the new high res data (We didn't 
 collect Zn peak data for the new crystal - didn't think we'd need it.). I 
 have done that and found 4 mols with Phaser in about 60 seconds. Still cool.

 So, we would like to transfer Free R flags to the new data to avoid refining 
 against what had been labelled as Free R.
 My problem is - how do I do that properly?
 I am worried that some of the working data in the bigger cell will be 
 correlated with Free data via the near crystallographic NCS.
 I clearly don't want to just copy them from the old mtz file with a0

 I recall some discussion about this from years ago on the BB but can't find 
 the right threads.

 Can anybody point me to the correct way to do this please - I presumably want 
 to label with Free R flags symmetry related Free R labelled reflections from 
 the old data that are related by the new NCS 2-fold (that is close to 
 crystallographic) in the new data. Right?? If I have worded that correctly...
 I am hoping that will make sense to somebody.

 I think that the solutions that were recently suggested for lower vs higher 
 symmetry in the same unit cell do not apply here.



 One suggestion has been to do the MolRep, choose new free Rs,  give it all a 
 good hard shake with high temp simulated annealing and hope that any bias is 
 gone.

 I'm not sure that I am comfortable with the word hope here...
 But, if the consensus of opinion of the wise folk at the BB is that this will 
 pass muster at the point where the charming and delightful referees are 
 commenting on the extremely high impact (obviously :-) manuscript, then I 
 will quote you all!


 I await your wise words.

 Free R. Again. Sorry.


 Cheers
 Ed


 __
 T.Edwards Ph.D.
 Garstang 8.53d
 Astbury Centre for Structural Molecular Biology
 University of Leeds, Leeds, LS2 9JT
 Telephone: 0113 343 3031
 http://www.bmb.leeds.ac.uk/staff/tae/
 

[ccp4bb] Free R with doubled cell edge

[Thomas Edwards]

Dear BB Sages,

I have a problem where I think I could very easily do the wrong thing.
And I don't really want to do that...

We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - 
Shelx, RESOLVE, ARPwARP. Cool.).
In p21 30 109 65 90 105 90 at 2.5A

However, we have now collected 1.9A data.
In p21...
60 109 65 90 107 90

4 chains per AU instead of 2 with a doubling of a.

Self rotations with the new data suggest 2 two-folds, one quite near 
crystallographic.
It seems that the doubling of the a edge is adding an NCS two-fold that is 
almost crystallographic.

Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would 
like to use that model to do MR against the new high res data (We didn't 
collect Zn peak data for the new crystal - didn't think we'd need it.). I 
have done that and found 4 mols with Phaser in about 60 seconds. Still cool.

So, we would like to transfer Free R flags to the new data to avoid refining 
against what had been labelled as Free R.
My problem is - how do I do that properly?
I am worried that some of the working data in the bigger cell will be 
correlated with Free data via the near crystallographic NCS.
I clearly don't want to just copy them from the old mtz file with a=30

I recall some discussion about this from years ago on the BB but can't find the 
right threads.

Can anybody point me to the correct way to do this please - I presumably want 
to label with Free R flags symmetry related Free R labelled reflections from 
the old data that are related by the new NCS 2-fold (that is close to 
crystallographic) in the new data. Right?? If I have worded that correctly...
I am hoping that will make sense to somebody.

I think that the solutions that were recently suggested for lower vs higher 
symmetry in the same unit cell do not apply here.



One suggestion has been to do the MolRep, choose new free Rs,  give it all a 
good hard shake with high temp simulated annealing and hope that any bias is 
gone.

I'm not sure that I am comfortable with the word hope here...
But, if the consensus of opinion of the wise folk at the BB is that this will 
pass muster at the point where the charming and delightful referees are 
commenting on the extremely high impact (obviously :-) manuscript, then I will 
quote you all!


I await your wise words.

Free R. Again. Sorry.


Cheers
Ed


__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
-- A new scientific truth does not triumph by convincing opponents and making 
them see the light, but rather because its opponents eventually die, and a new 
generation grows up that is familiar with it.  ~Max Planck

[ccp4bb] Postdoctoral fellowship available at the University of Leeds

[Thomas Edwards]

A Wellcome Trust funded post-doctoral position is available from September in 
the Astbury Centre for Structural Molecular Biology at the University of Leeds 
in the laboratories of Dr John Barr and Dr T. Edwards. The successful applicant 
will study structural and functional aspects of bunyavirus gene expression. We 
are looking for highly motivated candidates that have a Ph.D. in structural 
biology with a strong research background in molecular biology and/or virology.
The deadline for formal applications is the end of August.

See details below and:
http://www.nature.com/naturejobs/science/jobs/153434-Research-Fellow

Cheers,
Ed
__
T.Edwards Ph.D.
Director, Macromolecular Crystallography Unit
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
-- Science is simply common sense at its best.  ~Thomas Huxley



Research Fellow
(Fixed-term for 36 months, starting from September 2010)

Project Title: Structural and functional analyses of the bunyavirus 
ribonucleoprotein complex

You will study structural and functional aspects of bunyavirus gene expression, 
in the laboratory of Dr. John Barr.

The post is funded by The Wellcome Trust, and will focus on the structural and 
functional analysis of the nucleocapsid (N) protein of Bunyamwera virus (BUNV), 
which is a segmented negative stranded RNA virus of the family Bunyaviridae. 
The N protein is a major structural component of the virus, which binds the 
viral genome to form the ribonucleoprotein (RNP), and this association is 
critical for both RNA synthesis and virus assembly. The project will follow on 
from our recent crystallization of the BUNV N protein, and will use X-ray 
crystallography to determine the high-resolution structure of the N protein. 
This information will then be used to guide an investigation of known N protein 
functions, assays for which are already established in our laboratories. These 
assays include: N protein oligomerization, N:RNA interaction, RNP formation, 
polymerase binding and RNA synthesis activity.

A PhD in structural biology is required, together with a strong research 
background in molecular biology and/or virology.

Informal enquiries to Dr. John N. Barr, email j.n.b...@leeds.ac.uk or Dr. Tom 
A. Edwards, email t.a.edwa...@leeds.ac.uk

Re: [ccp4bb] control of nucleation

[Thomas Edwards]

Dear Zq,

A few ideas:

1) Vary protein concentration, temperature, or protein : mother liquor ratio.
2) Try dioxane - it is supposed to reduce nucleation.
3) give your protein a good hard spin before you set up drops to remove 
aggregates.
4) seeded factorial screen.
5) re-purify on gel filtration?

Ed

__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
-- Nature composes some of her loveliest poems for the microscope and the 
telescope.  ~Theodore Roszak




From: zq deng dengzq1...@gmail.com
Reply-To: zq deng dengzq1...@gmail.com
Date: Thu, 6 May 2010 09:03:46 +0100
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] control of nucleation

hello,everybody . due to excess nucleation,I often get many tiny crystals 
instead of  few,large crystals.i wana optimize the condition, does anyone have 
adivce about this?

Best regards.

[ccp4bb] ERC funded Project Studentship in Structural Molecular Biology

[Thomas Edwards]

Dear Crystallographers...

We have an EU funded studentship to study α-Helix Mimetics as Inhibitors of 
Protein-Protein Interactions Involved in Cancer Development and Progression.
Please forward to anybody who may be interested.

The purpose of this project is to develop a RULE-BASED APPROACH for the design 
and synthesis of inhibitors of key protein-protein interactions (PPIs) involved 
in the development and progression of cancer. The project is concerned with the 
analysis of a series of scaffolds designed to act as *-helix mimetics. These 
scaffolds are amenable to high-throughput solid-phase synthesis and thus the 
generation of libraries. We have recently shown that these scaffolds
can be functionalised such that they inhibit the p53-hDM2 interaction (an 
oncogenic target) with high
(*M affinity). The student will develop cell based assays to test novel 
aromatic oligoamides as
proteomimetic inhibitors of three cancer targets: p53/hDM2, Bcl-xL/BAK and 
Mcl-1/NOXA-B. The
student will also perform detailed biophysical analysis and structural studies 
including X-ray
crystallography, NMR and isothermal titration calorimetry on the inhibitors and 
their interactions with the
target protein. Inhibitors of the hDM2/p53 interaction are being sought to 
decrease tumour growth, with
potential clinical applications in a broad range of cancers, including breast 
cancer (more than half of
human cancers express a mutant p53). Bcl-xL and Mcl-1 are members of the Bcl-2 
family of proteins
and have also been proposed to be a means of intervention against various 
cancers including prostate
cancer and small cell lung carcinoma. This multidisciplinary project will 
provide opportunities for the
student to receive training in molecular biology, structural biology, molecular 
modelling and biophysical
analysis.

For details, and how to apply, please see:
http://www.bmb.leeds.ac.uk/staff/tae/ERC_Studentship.pdf

Cheers
Ed

__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
Science is always wrong. It never solves a problem without creating ten more. 
~George Bernard Shaw

[ccp4bb] Yorkshire Cancer Research funded studentship, University of Leeds

[Thomas Edwards]

Dear BB,

We have a funded PhD studentship position that we must fill with some urgency 
(the grant must start by 1st Feb 2010).
Please see details below, and this link:
http://www.bmb.leeds.ac.uk/staff/tae/YCR_advert.pdf

This is a collaboration between the Department of Chemistry and the Faculty of 
Biological Sciences at the University of Leeds. There is quite alot of 
flexibility in which direction the student may wish to push the project from 
synthetic chemistry to experiment structural biology to modelling, and we are 
therefore open to applications from students with a broad range of backgrounds.

I must, unfortunately, stress that funding body requirements state that this 
position is only open to UK/EU students.

I am happy to field informal enquiries at first, but a formal application must 
then be made.

Cheers
Ed
__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
Research is what I'm doing when I don't know what I'm doing. ~Wernher Von Braun



Development of Synthetic α -Helix Mimetics as Potent Anticancer Agents

Yorkshire Cancer Research funded Project Studentship

Dr A. Wilson, Dr T. Edwards and Dr R. Jackson. University of Leeds

The purpose of this project is to develop a RULE-BASED APPROACH for the
design and synthesis of inhibitors of key protein-protein interactions (PPIs)
involved in the development and progression of cancer.

The project is concerned with the development of a series of scaffolds
designed to act as α-helix mimetics. These scaffolds are
amenable to high-throughput solidphase synthesis and thus the generation
of libraries. We have recently shown that these scaffolds can be functionalised
such that they inhibit the p53-hDM2 interaction (an oncogenic target) with
high (μM affinity). The student will develop aromatic oligoamides as
proteomimetic inhibitors of three cancer targets: p53/hDM2, Bcl-xL/BAK and Mcl-
1/NOXA-B. Inhibitors of the hDM2/p53 interaction are being sought to decrease
tumour growth, with potential clinical applications in a broad range of cancers,
including breast cancer (more than half of human cancers express a mutant
p53). Bcl-xL and Mcl-1 are members of the Bcl-2 family of proteins and have
also been proposed to be a means of intervention against various cancers
including prostate cancer and small cell lung carcinoma. The goals of this
project are i) to optimise small molecules that mimic these α-helical
sequences and bind with high affinity and selectivity to their target protein,
inhibiting the native PPI; ii) use structural and molecular biology and
biophysics to understand what discriminates a high affinity ligand from one
with low affinity. This will provide a starting point for drug development. This
multidisciplinary project will provide opportunities for the student to receive
training in organic synthesis in addition to molecular biology, crystallography
and NMR, molecular modelling and biophysical analyses.

The stipend would be at the standard EPSRC rate (~ £13290 pa for 2009/10).
Applications for this 3 year studentship should be directed to Tanya
Wainwright, School of Chemistry, University of Leeds, Leeds, LS2 9JT and
can be made online via the following link:
http://www.chem.leeds.ac.uk/newRPG/RPGhowToApply.htm
(Please note: only EU applicants can be considered for this award)

Please contact Dr. T. Edwards (t.a.edwa...@leeds.ac.uk) or Dr. A. Wilson
(a.j.wil...@leeds.ac.uk) for further details about this project.

Re: [ccp4bb] nad woes

[Thomas Edwards]

Rob, Thanks for the tips of wisdom. Is the 3 letter code for NADH something different, or is it NAD as well? Ditto for FMN and its reduced form.Thomas Edwards-CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote: -To: CCP4BB@JISCMAIL.AC.UKFrom: Rob Meijers ccp4spama...@yahoo.comSent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UKDate: 03/19/2009 01:01AMSubject: Re: [ccp4bb] nad woesHi Jan, at low occupancy (and I suppose a resolution that does not extend beyond 2.0 A), you have to rely on your restraints. I think the consensus is that the adenine ring should be planar. The pyridine ring of the nicotinamide should be flat if the ring is oxidized (NAD+), and can be distorted when reduced (NADH). Most NAD molecules in the PDB have strict planar restaints in the pyridine ring of the nicotinamide, even when the density clearly shows that they are puckered. Many NAD+ cofactors get converted to NADH during crystallization by the enzyme they bind. Especially PEG can contain a substrates to convert NAD+ into NADH in the crystal. Best regards, Rob Meijers Synchrotron Soleil --- On Thu, 3/19/09, Jan Abendroth jan.abendr...@gmail.com wrote: From: Jan Abendroth jan.abendr...@gmail.com Subject: [ccp4bb] nad woes To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, March 19, 2009, 12:50 AM Hi all,is there any wisdom on NAD out there? I experience some strange behaviour ofthis common cofactor.With moderately convincing density, probably low occupancy of a cofactor thatcame along for the ride from E coli, Refmac5.5.0088 pulls the AN6 atom out ofthe adenine plane. With my own library that puts planar restraints on theadenine ring this seems to be fixed.Coot during real space refinement or regularisation using either the standardor my own dictionary handles the purine ring just fine, however, totally garblesup the nicotineamide.Btw, I use the * nomenclature.CheersJan--Jan AbendrothdeCODE biostructuresSeattle /Bainbridge Island WA, USAwork: JAbendroth_at_decode.ishome: Jan.Abendroth_at_gmail.com

[ccp4bb] phased MR

[Thomas Edwards]

Dear BB,

 

I would like to ask for some advice on phased molecular replacement if
possible.

 

I have a MR model that has so far not proved successful with Phaser,
Molrep, Amore, Beast etc.

 

I have SeMet MAD data to 3.6A that gives decent looking anomalous
difference peaks, looks stable in mlphare, and produces solvent
flattened maps to 2.8A in DM that look like the density might be a
sensible shape - wrt solvent gaps etc - but not interpretable so far (I
will be trying phasing in SHARP, CNS etc).

In the mean time, is there a good way to combine phases that may be
slightly sensible with molecular replacement? Things may also be
complicated by the possibility of NCS translations...

 

Any advice gratefully received.

 

Many thanks to all those who continue to provide excellent suggestions,

Cheers

Ed

 

__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology 
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
http://www.bmb.leeds.ac.uk/staff/tae/Research
http://www.bmb.leeds.ac.uk/staff/tae/
If you're not part of the solution, you're part of the precipitate.
~Henry J. Tillman

 

[ccp4bb] DLS options?

[Thomas Edwards]

 

Dear BB,

 

Sorry for the off topic question:

 

I would like to buy a Dynamic Light Scattering system.

Could people suggest which they like the best and/or which is best
value?

 

I have in the past used a Protein Solutions Dyna Pro with micro cuvette
(I would like a micro cuvette option). However, it was very sensitive to
dust and bubbles, and the cuvette collects dust...

I've never tried the one from Precision Detectors.

Any other options?

 

Thanks

Ed

 

__
T.Edwards Ph.D.
Garstang 8.53d
Astbury Centre for Structural Molecular Biology 
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
http://www.bmb.leeds.ac.uk/staff/tae/
http://www.bmb.leeds.ac.uk/staff/tae/Research 
The doubter is a true man of science; he doubts only himself and his
interpretations, but he believes in science. ~Claude Bernard

 

[ccp4bb] imosflm gets stuck - Mac OS 10.5.2

[Thomas Edwards]

Dear BB,

Trying to install imosflm on my new Mac Pro running OS 10.5.2

imosflm will load the images, identify spots on the first frame, says it
is trying to find spots on the last frame but then goes into spinning
beachball mode and nothing happens. There is some data written out to
mosflm.lp but it looks like writing to that file hung half way along a
line too.
Any suggestions gratefully received.

Many thanks as ever.
Ed

__
T.Edwards Ph.D.
Astbury Centre for Structural Molecular Biology 
University of Leeds, Leeds, LS2 9JT  
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/
If you're not part of the solution, you're part of the precipitate.
~Henry J. Tillman

Re: [ccp4bb] imosflm gets stuck - Mac OS 10.5.2 - solved.

[Thomas Edwards]

Dear Harry,

problem solved.
You are a gentleman, sir!

Thanks
Ed


-Original Message-
From: Harry Powell [mailto:[EMAIL PROTECTED]
Sent: Mon 4/14/2008 5:57 PM
To: Thomas Edwards
Cc: [EMAIL PROTECTED]
Subject: Re: [ccp4bb] imosflm gets stuck - Mac OS 10.5.2
 
Hi Ed

just a thought - was Mosflm (not iMosflm) built with g77 or gfortran  
on your machine? If it was built with gfortran, you will probably have  
to set the environment variable  GFORTRAN_UNBUFFERED_ALL to 1 or y -  
easiest to do in your .cshrc (if you are running under tcsh or csh)  
or .bashrc (if you are running under bash) -

tcsh/csh -

setenv  GFORTRAN_UNBUFFERED_ALL 1

bash/zsh

export  GFORTRAN_UNBUFFERED_ALL=1

HTH,

On 14 Apr 2008, at 15:56, Thomas Edwards wrote:

 Dear BB,

 Trying to install imosflm on my new Mac Pro running OS 10.5.2

 imosflm will load the images, identify spots on the first frame,  
 says it is trying to find spots on the last frame but then goes into  
 spinning beachball mode and nothing happens. There is some data  
 written out to mosflm.lp but it looks like writing to that file hung  
 half way along a line too.

 Any suggestions gratefully received.

 Many thanks as ever.
 Ed

 __
 T.Edwards Ph.D.
 Astbury Centre for Structural Molecular Biology
 University of Leeds, Leeds, LS2 9JT
 Telephone: 0113 343 3031
 http://www.bmb.leeds.ac.uk/staff/tae/
 If you're not part of the solution, you're part of the precipitate.   
 ~Henry J. Tillman


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

Re: [ccp4bb] MolRep of coiled coils

[Thomas Edwards]

Sorry - I should have added that, yes, there are 2 identical peptide chains 
that should be parallel coiled-coil.
Any advice gratefully received.
Ed


-Original Message-
From: cockburn [mailto:[EMAIL PROTECTED]
Sent: Fri 3/28/2008 1:35 PM
To: Thomas Edwards
Subject: Re: [ccp4bb] MolRep of coiled coils
 
Hi Thomas,
Does your coiled-coil consist of two polypeptide chains, and are their 
sequences identical? Do you expect it to be a parallel or anti-parallel 
coiled-coil?
Yours
Joe

Thomas Edwards a écrit :
 Dear BB,

 I am attempting molecular replacement with a 2.8A data set from crystals of a 
 coiled coil of about 150 residues.
 Probably p21212 but maybe p2221.

 So far, Phaser, MolRep, Amore, Mr Bump, have not provided a good solution as 
 judged by Z-scores, CCs, Rfactors, and whether there is any density outside 
 the model (there should be - I'm using a slightly shorter model to search 
 with).

 One possible problem is that the coiled coil may not be straight. It may have 
 a slight curve to it.
 I have used models from the PDB that are straight or slightly curved, so far 
 with no success.
 I have tried a few different resolution cutoffs too.
 I have tried single helix chains or dimeric coiled coils.

 Is there anything special about MR with coiled coils?
 Can anybody provide any tips/hints on MR with coiled coils??

 Thanks
 Ed
   

[ccp4bb] FW: polarrfn, pltdev - problem solved

[Thomas Edwards]

Thanks to Ian and Marcus.
However, it turned out not to be a pltdev problem at all...

It looked like polarrfn was generating maps OK, but I guess not.
When I ran the same thing on a Mac OSX everything worked.
Great.
I have some peaks. Now I need to think. Hmmm.

Not sure what the problem was - other CCP4 things are running fine on the Suse 
box.
For the moment, I have peaks and I guess I will ignore the problem and happily, 
blindly, carry on...

Thanks
Ed


-Original Message-
From: Ian Tickle [mailto:[EMAIL PROTECTED]
Sent: Thu 3/6/2008 1:34 PM
To: Thomas Edwards
Cc: ccp4bb
Subject: RE: polarrfn, pltdev
 

Hi Thomas

I normally run pltdev on the whole map, did you try that?  There may be
a problem just plotting one section because I think it may rescale the
SRF values to the origin value on the first section, so if that happens
to be zero on the kappa=180 section you'll get division by zero.  The
origin peak on the kappa=0 section cannot be zero!

BTW I would recommend using E's instead of sharpening (via ECALC), since
your B = -20 is completely arbitrary and may not be sufficient.  Also
you shouldn't specify resolution cutoffs at all, the low res cutoff is
only needed if you don't have sharpening of any kind (otherwise the big
F's at low res dominate).  The high res cutoff isn't needed either
because the SRF isn't like the cross-RF where differences in the model
will give errors at high res.  For the SRF there is no model!
Sharpening  using all data helps to resolve peaks if the NCS is
complicated or is close to the space group peaks (of course if you're
only expecting 1 peak well separated from the space group peaks, it's
not likely to affect to your conclusions).  Finally having the radius
cutoff too small may reduce the signal/noise ratio (both signal and
noise increase with increasing R, the question is which one increases
faster).  I would suggest you try R=25 and R=30 as well as R=20 and see
what happens.

HTH

Cheers

-- Ian

 -Original Message-
 From: [EMAIL PROTECTED] 
 [mailto:[EMAIL PROTECTED] On Behalf Of Thomas Edwards
 Sent: 06 March 2008 12:42
 To: ccp4bb
 Subject: polarrfn, pltdev
 
 Dear CCP4BB,
 
 I'm trying to genereate some self rotations with polarrfn to 
 decide on the NCS and number of molecules in my AU.
 
 using polarrfn script (copied from the examples on the web):
 
 #
 #  Self-rotation function
 #
 set resolution = 15 3.5
 set radius = 20
 
 #  Calculate whole map,  search for peaks
 polarrfn hklin p2221_33A mapout p2221_self  eof-1
 title p2221 Self-Rotation function, resolution ${resolution} 
 radius ${radius}
 self  ${radius}
 resolution  ${resolution}
 crystal  file 1 bfac -20
 labin  file 1  F=F SIGF=SIGF
 map
 find  5 100
 eof-1
 
 # Now plot just the kappa = 180 deg section
 plot:
 polarrfn hklin p2221_33A mapin p2221_self plot p2221_self  eof-2
 title p2221 Self-Rotation function, resolution ${resolution} 
 radius ${radius}
 self  ${radius}
 resolution  ${resolution}
 crystal  file 1 bfac -20
 labin  file 1  F=F SIGF=SIGF
 read 180 180
 plot  10 10
 eof-2
 
 
 As you can guess, I think I'm in p2221... The script seems to 
 run fine and spits out map and .plo files. 
 However, when I try to run pltdev to generate a postscript 
 file (this used to work in Unix in a previous lab...)
  pltdev -i p2221_self.plo
 I get a file (plot84.ps) with lots of nan records (see below).
 I'm running Suse 10.3, CCP4 6.0.2
 Any pointers in the right direction much appreciated.
 
 Thanks
 Ed
 
 
 %!PS-Adobe-3.0
 %%Creator: pltdev
 %%Pages: 1
 %%BoundingBox: -2147483648 -2147483648 -2147483648 -2147483648
 %%EndComments
 %%BeginProlog
 /L {lineto} bind def
 /M {moveto} bind def
 /S {stroke} bind def
 /N {newpath} bind def
 %%EndProlog
 %%Page: 1 1
 %%PageBoundingBox: -2147483648 -2147483648 -2147483648 -2147483648
 0.8 setlinewidth 1 setlinejoin N 0 0 M
 S N nan nan M
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 nan nan L
 
 


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[ccp4bb] Post-doctoral position Edwards lab University of Leeds

[Thomas Edwards]

 Picture (Metafile) 
Faculty of Biological Sciences
Institute of Molecular  Cellular Biology
Astbury Centre for Structural Molecular Biology

Research Fellow: University Grade 6/7 (£21,682 - £26,666 p.a)
Project Title: The Structure of Dazl based translation control complexes
Job ref 313168 Closing date 15/6/07

We are seeking outstanding and motivated candidates for a postdoctoral position 
in the laboratory of Dr Thomas Edwards within the Astbury Centre for Structural 
Molecular Biology at the University of Leeds. The focus of the project is the 
structure and function of proteins that control translation from mRNAs during 
germ cell development. Crystallographic studies will be used to investigate the 
RNA binding and translation control properties of DAZ family proteins and 
associated cofactors. Molecular biology, protein expression and purification, 
and X-ray crystallography will be required to pursue this project.
 
The Fellow's research will benefit from the outstanding resources in the 
Astbury Centre for Structural Molecular Biology at Leeds. The X-ray facilities 
include an Raxis IV, with Osmic mirrors and Oxford cryostream, on a Rigaku 
rotating copper anode X-ray generator. Additionally, there will be regular 
synchrotron access at Daresbury, DIAMOND and Grenoble. A state-of-the-art 
robotic crystallization facility includes liquid handling and nano-liter 
drop-setting. Protein expression and purification facilities include tissue 
culture, facilities for bacterial and insect cell growth, and Åkta purification 
systems.
 
Leeds is an exciting multi-cultural city that is the fastest growing in 
England. It is unrivalled in the north of England as a major shopping 
destination and centre for the arts, entertainment, nightlife and leisure. The 
city has a rich cultural heritage with a wide range of theatres, cinemas, 
museums and art galleries and a thriving music scene to cater for all tastes.  
Leeds has a proud sporting tradition and there are plenty of opportunities to 
participate at all levels. Nature lovers will enjoy the nearby Yorkshire Moors 
and Dales and find other outdoor recreation easily accessible.

For details of the Edwards lab see http://www.bmb.leeds.ac.uk/staff/tae/. 
Please direct informal enquiries to [EMAIL PROTECTED] To apply online please 
visit http://www.leeds.ac.uk/about/jobs/ and click on jobs.  Alternatively, 
application packs are available from Mr A Bateman, Faculty Staff Recruitment 
Office, tel 0113 343 8040, email [EMAIL PROTECTED]



__
T.Edwards Ph.D.
Astbury Centre for Structural Molecular Biology 
University of Leeds, Leeds, LS2 9JT  
Telephone: 0113 343 3031
http://www.bmb.leeds.ac.uk/staff/tae/

ole0.bmp

Re: [ccp4bb] Unusual Difference Fourier near Methionines

[Thomas Edwards]

 
Dear Ethayathulla ,
 
did you solve the structure by SeMet MAD or some other way?
Sometimes you get strange densities around the ends of methionine side
chains (around the S/Se) when switching from SeMet data to native.
 
Cheers
Ed
 

__ 
T.Edwards Ph.D. 
Astbury Centre for Structural Molecular Biology 
University of Leeds, Leeds, LS2 9JT  
Telephone: 0113 343 3031 

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
Behalf Of Ethayathulla Abdulsamath
Sent: Fri 13 Apr 2007 10:51
To: [EMAIL PROTECTED]
Subject: [ccp4bb] Unusual Difference Fourier near Methionines




Dear all

I am doing one structure at 2.6A resolution where I found
unusual density near methionines. Actually three methionines come close
together nearby and I get difference even at 5sigma cutoff. I don't
understand nature of the density. The amino acid sequnce is same I mean
met by chemical sequncing so there is no change amino acid residues. I
am sending the snapshot of the difference fourier observed at
methionines. The crystal belong to R32 system with one molecule and
biological trimer. In snapshot two of the methionines are symmetry
related. 

Details of the data collection. 
Data collected at synchrotron

Overall Rsym = 6 % and I/sigma = 2.1. Completeness = 99% 
  
Can anyone suggestion what could be chemical nature for the
difference fourier.

Thanks in advance

Ethayathulla 

### 
A.S.Ethayathulla,Ph.D. 
Department of Biophysics 
All India Institute of Medical Sciences 
Ansari Nagar 
New Delhi-110029 
India. 
### 



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