Re: [ccp4bb] Engineering Recombinant Proteins: Biochem Soc Training day, Nov 4-5, 2019

[Evans, Nicola]

Dear All,


A reminder that the earlybird deadline for the course on Protein engineering by 
the Biochemical Society is coming up on the 15th May.


For more information the link is here:

https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA219/view/Conference/Default.aspx

Protein engineering II: from new molecules to new processes
15—17 July 2019

University of York, UK


The ability to engineer proteins to tackle new challenges has had a 
transformational effect across the biological and physical sciences, in both 
industrial and academic settings. The rate at which new methods are being 
developed shows no sign of slowing. This timely meeting will cover recent 
developments in the engineering of proteins. Themes include new methods such as 
computational design, high throughput screening, and genetic code expansion, 
targets such as new biotherapeutics, scaffolds and catalysts, and the 
applications to which these are being applied, including diagnostics and 
sensing, nanotechnology, and synthetic biology. As such this will be an 
interdisciplinary meeting that will demonstrate the state-of-the-art of the 
field, and will bring together a diverse set of researchers, making this an 
ideal forum for the exchange of new ideas.



Oral communication slots are available at this meeting. All attendees, 
particularly researchers in the early stages of their career, are invited to 
submit a poster abstract for consideration as an oral communication.



Programme Coordinators:

Rivka Isaacson, King's College London

Andrew Thomson, University of Glasgow

Dafydd Jones, Cardiff University

Paul Ko Ferrigno, Metalinear



From: CCP4 bulletin board  on behalf of Amir Khan 

Sent: 08 April 2019 14:37:00
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Engineering Recombinant Proteins: Biochem Soc Training day, 
Nov 4-5, 2019

Hi,

This is a ‘save-the-date’ message for an upcoming ‘Training Day’ organized by 
the
Biochemical Society.  The event will be held in London (UK) and is entitled:

‘Engineering recombinant proteins for structural and functional studies’

The programme will address the limiting step in structural studies - the 
production of
pure and homogenous proteins.  Talks and discussions at this event will explore 
strategies
to optimize membrane and soluble proteins for crystallization, cryo-EM and NMR 
studies.
Discussions will include construct optimization, novel thermostability assays,
crystallization tools and ‘tricks of the trade’ to enable structure 
determination.

This event is particlarly directed toward early-stage graduate students in 
structural biology.
However, anyone interested in generating soluble proteins for a variety of 
applications,
such as enzymatic assays and antibody production, would be welcome to attend.
Manufacturers of instruments and techniques to assay purity and homogeneity
will be available for ‘hands-on’ demonstrations.

The list of invited speakers are experienced in the structure determination of
membrane proteins, cytosolic/secreted proteins, and macromolecular signaling 
complexes:

Edmund Kunji, Cambridge
Daniel Panne, Leicester
Maria Sasi Conte, King’s College
Naomi Chayen, Imperal College
Laura Itzhaki, Cambridge
Chris Tate, Cambridge

More details about the programme and venue will be provided in the coming weeks.

Best wishes,

Amir Khan, Trinity College Dublin
Rivka Isaacson, King’s College




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Re: [ccp4bb] LAST CHANCE: DLS/CCP4 data analysis workshop 2018

[Evans, Nicola]

I did this course last year and I would highly recommend it! Also it is not 
just for PhD students or early postdocs, there were a few of us last year with 
more experience and we also got a lot out of it, especially in the practical 
data collection and processing part (in the safe hands of computational 
experts).


Nicola


From: CCP4 bulletin board  on behalf of David Waterman 

Sent: 03 October 2018 21:19:39
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] LAST CHANCE: DLS/CCP4 data analysis workshop 2018

Dear all,

The application period for this year's DLS/CCP4 workshop closes this Sunday. 
Please get your applications in soon if you would like the opportunity to 
collect data at the UK's national synchrotron light source, and get help from 
leading experts on your own projects.

Best wishes
-- David


On Wed, 19 Sep 2018 at 14:17, David Waterman 
mailto:dgwater...@gmail.com>> wrote:
PhD students, postdocs and early career scientists,

Please consider applying for the fifth joint DLS/CCP4 workshop on MX data 
collection and structure solution, to be held at Diamond Light Source, UK from 
the 2nd to the 9th December (with accommodation from the 1st).

This course offers the opportunity for you to work alongside leaders in the 
field of MX on data from your own crystals. For more details please check here:

http://www.ccp4.ac.uk/schools/DLS-2018/

The application period is now open and will close on the 7th October. 
Applicants will be required to submit a CV/resume, describe their projects and 
obtain a letter of support from a supervisor, so please do not wait until the 
last minute to apply! Those with crystals and/or data will be given priority.

Best wishes on behalf of the organisers,

David Waterman (CCP4)




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Re: [ccp4bb] crystallization optimization

[Evans, Nicola]

All great suggestions and a few new ones I haven't used before (thanks Emmanuel 
and Frank).


I agree with Emmanuel, a fine screen should definitely be first port of call, 
change the precipitant to go higher and lower than the initial hit (I normally 
vary the PEG in 2% jumps, in a 24 or 48 well screen), and also the pH by 0.5-1 
points above and below. I would also try different temperatures (higher & 
lower), and different protein:well ratios (1:1, 1:2, 2:1).


Seeding is also my favourite method for optimisation, I usually seed into my 
normal fine screen (with a 1:2:2, seed:protein:well ratio, if working from an 
initial 1:1 ratio), and sometimes a new screen with greatly reduced 
precipitant, but the former has always worked better in my microcrystal cases.


There are a lot of suggestions for reducing the protein concentration, I agree, 
but just to note I once had a case where just diluting the protein with buffer 
didn't help as the nucleation points didn't break down (tried to reduce 8mg/ml 
to 4 and 2mg/ml). Instead we did a fresh prep and made sure the concentration 
didn't go above 4mg/ml throughout the whole prep, and this worked for us (with 
a 2:1 protein:well ratio).


Are there any cysteines in the protein? Sometimes adding more reducing agents 
helps reduce nucleation. Also sometimes adding a ligand can help improve 
protein crystal size and quality.


Good luck, let us know if anything works!


Nicola








From: CCP4 bulletin board  on behalf of Emmanuel 
Saridakis 
Sent: 12 July 2017 13:55:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Dear All,

Fine-tuning protein and precipitant concentration is of course the first line 
of approach, followed by both rMMS and streak-seeding.

I would like to remind you of a far less popular but often successful in my 
hands, optimisation technique: It consists in incubating the trial at the 
condition that gives the ugly crystals for some time (shorter than the time it 
takes for the ugly crystals to appear), and then changing the condition to one 
of lower supersaturation for the growth to proceed. That can be done either by 
diluting the reservoir with water (vapour diffusion) or diluting the drop with 
buffer (microbatch), or by just transferring your plate to a different (usually 
higher) temperature.

Described in more (but perhaps unecessary for most practical purposes) detail 
in:
E. Saridakis, P.D. Shaw Stewart, L.F. Lloyd and D.M. Blow. Acta Crystallogr. 
(1994) D50, 293.
E  E. Saridakis and N.E. Chayen. Protein Science (2000) 9, 755.

Best,
Emmanuel


De: "Alun R Coker" 
À: "CCP4BB" 
Envoyé: Mercredi 12 Juillet 2017 15:40:10
Objet: Re: [ccp4bb] crystallization optimization


Hi Everyone,

Franks point is really interesting. We routinely reduce the protein 
concentration when we see too many precipitated wells, but we never dilute the 
screen. Has anyone tried this?

All the best,

Alun

On 12/07/17 08:48, Frank von Delft wrote:

The point I was failing to make:  reducing either protein or precipitant 
concentration will indeed reduce nucleation, but often won't get you bigger or 
more single crystals:  it will just make the appearance of crystals less 
reliable.

The way to get big single reliable crystals is to increase protein and greatly 
reduce precipitant.

(Even better:  do seeding.  Like Vicky said.  Incredible how often people don't 
bother to do seeding, yet it solves so many problems.)

phx


On 12/07/2017 07:50, Vicky Tsirkone wrote:
Dear Frank,

I may see in the attached pic several nucleation points and a considerable 
amount of microcrystals. Based to my knowledge decreasing the concentration of 
the precipitant and/or the protein concentration would be a reasonable approach 
to refine the initial hits.
By checking the diagram as you correctly mentioned you may see that the fine 
tuning of protein and precipitant concetration may lead to the desirable result 
without reaching the precipitation zone.

Patrick just check your screens. Just a rule of thumb, if you see precipitation 
in the ~60% of your drops then you should definitely reduce the protein 
concentration.

ps dont forget to try the streak seeding, as well.

Have a nice day and again good luck.

Vicky

On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft 
> wrote:

Actually, you should try increasing the protein concentration - a lot.  But be 
prepared to drop the precipitant concentration to almost nothing (1 or 2% isn't 
"low").

To understand why, look at the phase diagram and what we assume about vapour 
diffusion.  (Which I'm assuming is what you're doing.)


On 12/07/2017 06:28, Vicky Tsirkone wrote:
Dear Patrick,

You may reduce the protein concentation, as well.
Another option could be the streak 

Re: [ccp4bb] Any suggestions?

[Evans, Nicola]

Yes no images came through, however on looking at your crystallisation 
conditions for clues, is there anything in the protein prep that could have 
been carried through, even from the early stage buffers, or cell growth media? 
We once had a detergent in the cell lysis stage that was removed from all 
subsequent buffers (and protein put down 2 columns) but it made it through and 
produced density in the crystal structure.


Good luck with your search,


Nicola


From: CCP4 bulletin board  on behalf of Ian Clifton 

Sent: 07 July 2017 14:23:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Any suggestions?

"Dr. Isabel  De Moraes"  writes:

> Any suggestions regarding to the positive densities?
>
Your pictures didn’t seem to make it as attachments, at least as
received here.

BW,
--
Ian ◎

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