Re: [ccp4bb] looking for paper describing optimisation of crystals using a screen kit as additive

[Birtley, James]

Sebastiano,



I have had really nice success by mixing initial crystallization hits back in 
to sparse matrix screen conditions. My personal favourite is using the Wizard 
screens.



http://www.rigakureagents.com/p-1-wizard-classic-crystallization-screen-series.aspx



I found 75% of the original condition mixed with 25% of a sparse matrix screen 
results in many drops containing crystals with different habits.

This allowed me to get around anisotropy and to improve the resolution from 
poorly diffracting crystals.



A primary reference for this is found here:


Acta Crystallogr D Biol 
Crystallogr. 2005 May;61(Pt 
5):646-50. Epub 2005 Apr 20.
Crystallization of foot-and-mouth disease virus 3C protease: surface 
mutagenesis and a novel crystal-optimization strategy.
Birtley JR1, Curry S.
 Good luck with your experiments.

James

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andreas Forster 
[docandr...@gmail.com]
Sent: 08 May 2017 09:57
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] looking for paper describing optimisation of crystals 
using a screen kit as additive

Hi Sebastian,

you're thinking about local sparse matrix screening.  I've done this at a 90:10 
ratio.

Majeed, S., Ofek, G., Belachew, A., Huang, C.C., Zhou, T., and Kwong, P.D.
Enhancing protein crystallization through precipitant synergy.
Structure. 2003; 11: 1061–1070

All best.


Andreas



On Mon, May 8, 2017 at 3:18 PM, Sebastiano Pasqualato 
> wrote:

Dear all,
I recall a paper (or was is a talk at a conference?) describing the 
optimisation of initial hits of crystallisation by using a standard screen kit 
as additive.
Something like setting the tray using the initial crystallisation hit condition 
in the reservoir and mixing 75% of the hit condition with 25% of a commercial 
sparse matrix screen kit with the protein in the drop.
I can’t find the reference, can anybody help me?
Thanks a lot,
ciao,
Sebastiano


--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit

Re: [ccp4bb] looking for paper describing optimisation of crystals using a screen kit as additive

[Andreas Forster]

Hi Sebastian,

you're thinking about local sparse matrix screening.  I've done this at a
90:10 ratio.

Majeed, S., Ofek, G., Belachew, A., Huang, C.C., Zhou, T., and Kwong, P.D.
Enhancing protein crystallization through precipitant synergy.
Structure. 2003; 11: 1061–1070

All best.


Andreas



On Mon, May 8, 2017 at 3:18 PM, Sebastiano Pasqualato <
sebastiano.pasqual...@gmail.com> wrote:

>
> Dear all,
> I recall a paper (or was is a talk at a conference?) describing the
> optimisation of initial hits of crystallisation by using a standard screen
> kit as additive.
> Something like setting the tray using the initial crystallisation hit
> condition in the reservoir and mixing 75% of the hit condition with 25% of
> a commercial sparse matrix screen kit with the protein in the drop.
> I can’t find the reference, can anybody help me?
> Thanks a lot,
> ciao,
> Sebastiano
>
>
> --
> *Sebastiano Pasqualato, PhD*
> Crystallography Unit
> Department of Experimental Oncology
> European Institute of Oncology
> IFOM-IEO Campus
> via Adamello, 16
> 20139 - Milano
> Italy
>
> tel +39 02 9437 5167 <+39%2002%209437%205167>
> fax +39 02 9437 5990 <+39%2002%209437%205990>
> web http://is.gd/IEOXtalUnit
>
>

[ccp4bb] looking for paper describing optimisation of crystals using a screen kit as additive

[Sebastiano Pasqualato]

Dear all,
I recall a paper (or was is a talk at a conference?) describing the 
optimisation of initial hits of crystallisation by using a standard screen kit 
as additive.
Something like setting the tray using the initial crystallisation hit condition 
in the reservoir and mixing 75% of the hit condition with 25% of a commercial 
sparse matrix screen kit with the protein in the drop.
I can’t find the reference, can anybody help me?
Thanks a lot,
ciao,
Sebastiano


-- 
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEOXtalUnit

Re: [ccp4bb] BSA as additive

[Keller, Jacob]

Regarding that paper, I would point out that cytosols generally contain 50-100 
mM glutamate, so it makes sense that glutamate enhances solubility.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Evans, 
Nicola
Sent: Monday, May 08, 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] BSA as additive


I haven't used BSA, but I did recently use 50mM L-glutamic acid for this exact 
reason (and 5% glycerol in all buffers except the last one for crystallography) 
after reading this paper and it made a big difference to my last protein prep: 
https://www.ncbi.nlm.nih.gov/pubmed/15264823



For my final crystallography buffer I have tried with and without L-glutamic 
acid (as I am trying to optimise micro-crystals and worried the L-glu would 
make sample too soluble) but still waiting to see if I get any improvement. 
Both have drops with micro-crystals already (after 2 days), the L-glutamic acid 
sample has fewer, hoping some other drops will yield better crystals over time.



Hope that helps!



Nicola


From: CCP4 bulletin board > 
on behalf of Ha Sin >
Sent: 08 May 2017 12:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] BSA as additive

[Evans, Nicola]

I haven't used BSA, but I did recently use 50mM L-glutamic acid for this exact 
reason (and 5% glycerol in all buffers except the last one for crystallography) 
after reading this paper and it made a big difference to my last protein prep: 
https://www.ncbi.nlm.nih.gov/pubmed/15264823


For my final crystallography buffer I have tried with and without L-glutamic 
acid (as I am trying to optimise micro-crystals and worried the L-glu would 
make sample too soluble) but still waiting to see if I get any improvement. 
Both have drops with micro-crystals already (after 2 days), the L-glutamic acid 
sample has fewer, hoping some other drops will yield better crystals over time.


Hope that helps!


Nicola


From: CCP4 bulletin board  on behalf of Ha Sin 

Sent: 08 May 2017 12:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] BSA as additive

[Briggs, David C]

(Sorry... over-enthusiastic sending)


As for an actual reference - I'm not sure.


It's a bit like lab folk-lore.


Dave


Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Ha Sin 

Sent: 08 May 2017 13:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] BSA as additive

[Briggs, David C]

Hello,


I've seen BSA used to "block" membranes of centrifugal concentrators when 
proteins were sticking to them.


It appeared to work (proteins could be concentrated with less loss), but I'm 
not sure how much BSA ends up in your sample.


I can imagine the answer is "a bit" and therefore for some techniques this 
might not be appropriate.


Dave


Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Ha Sin 

Sent: 08 May 2017 13:32:44
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] BSA as additive

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a reference 
where BSA has been used as an "additive" in the protein concentration step to 
prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

[ccp4bb] BSA as additive

[Ha Sin]

Dear CCP4BB members,

Sorry about my non crystallography question. Does anyone know of a
reference where BSA has been used as an "additive" in the protein
concentration step to prevent aggregation of the main protein?

I would appreciate your suggestions.

Thank you,

H.Sin

Re: [ccp4bb] how to run shelx C/D/E using CCP4 GUI on windows

[Eleanor Dodson]

I cant say anything about Windowssystems - jobs are MEANT to run on either.

But re Intensities - you will have intensities in an mtz file somewhere -
all data processing outputs both amplitudes and intensities..

As a rule the conversion for I to F and back does not change the stronger
intensities much and again as a rule, the SHELX chain will work. However
the intermediate conversions are best avoided if you still have intensities
available
Eleanor

On 8 May 2017 at 10:57, chen c  wrote:

> Hi everyone,
>
> Can anybody tell how to run shelx C/D/E within CCP4 GUI on windows system?
> Moreover, since shelx C/D/E within CCP4 using mtz file (structure factor)
> instead of sca file (intensity), would this matters in tough conditions?
>
> Thank you!
>
> Best regards
> Chen
>
>
>
>
> --
> Cheng Chen, Ph.D. Candidate
> Laboratory of Structural Biology
> Life Science Building,Tsinghua University
> Beijing 100084
> China
> Tel:+86-10-62772291 <+86%2010%206277%202291>
> Fax:+86-10-62773145 <+86%2010%206277%203145>
> E-mail:che...@xtal.tsinghua.edu.cn
>
> 北京市海淀区清华大学生命科学馆201-212室
> 邮编：100084
>

[ccp4bb] how to run shelx C/D/E using CCP4 GUI on windows

[chen c]

Hi everyone,

Can anybody tell how to run shelx C/D/E within CCP4 GUI on windows system?
Moreover, since shelx C/D/E within CCP4 using mtz file (structure factor)
instead of sca file (intensity), would this matters in tough conditions?

Thank you!

Best regards
Chen




-- 
Cheng Chen, Ph.D. Candidate
Laboratory of Structural Biology
Life Science Building,Tsinghua University
Beijing 100084
China
Tel:+86-10-62772291
Fax:+86-10-62773145
E-mail:che...@xtal.tsinghua.edu.cn

北京市海淀区清华大学生命科学馆201-212室
邮编：100084

[ccp4bb] Postdoc (Structural Cell Biology) on targeting E3 ubiquitin ligases at Dundee

[Dr Alessio Ciulli]

Job title: Postdoctoral Research Assistant (E3 ubiquitin ligases)
Job location: Dundee, UK
Job closes: 21-May-2017

Professor Alessio Ciulli’s laboratory is seeking a postdoctoral researcher to 
undertake research in collaboration with one of the leading pharmaceutical 
companies supporting the Division of Signal Transduction Therapy (DSTT). The 
DSTT facilitates collaborations between some of the world’s major 
pharmaceutical companies (Boehringer Ingelheim, GlaxoSmithKline, and 
Merck-Serono) and leading Dundee-based researchers with a proven track record 
of achievement in understanding and exploiting the fundamental molecular causes 
of human disease in the fields of cancer, immune-regulation, neurodegeneration 
and hypertension resulting from disruptions in protein ubiquitylation, 
phosphorylation and other signalling networks.

The successful applicant will be based within the Ciulli lab in Dundee. The 
laboratory is pioneering novel chemical structural biology approaches to target 
E3 ligases (see Lucas and Ciulli, Curr. Opin Struct Biol 2017 and Gadd et al. 
Nat. Chem Biol. 2017 for recent publications from the group in this area). The 
successful applicant is expected to employ state-of-the-art structural biology, 
cell biology and biophysical approaches to conduct molecular and structural 
studies on disease-relevant human E3 ubiquitin ligases. 

The post is available immediately and salary will depend on experience, but 
will be up to £38,183. 
This is a unique opportunity to gain experience in industry-style drug 
discovery within an academic setting.

The position is available for 2 years with possibility of further 2 years 
extension.

Further details about the position and how to apply please visit
https://goo.gl/5FhYyW  


-
Alessio Ciulli, FRSC
Professor of Chemical & Structural Biology
School of Life Sciences, University of Dundee
Division of Biological Chemistry and Drug Discovery
James Black Centre, Dow Street, Dundee DD1 5EH
United Kingdom

Phone: +44 (0)1382 386230Fax: +44 (0)1382 386373
http://www.lifesci.dundee.ac.uk/groups/alessio-ciulli