Re: [ccp4bb] Phasing with Many Monomers/AU

[Steiner, Roberto]

In my experience translational NCS also can a part when one has many molecules 
in the a.u. If MR is an option, modern packages are rather good in dealing with 
TNCS.
We used Molrep + Refmac at 3.x A (still unpublished) for a case with 18 
complexes (36 monomers) in the a.u. and things weren't as bad as I originally 
thought.

BTW, we later made the construct 3 aa longer and we got 1 dimer in the a.u. 
with xtals diffracting to 2 A. I would consider playing a bit with your 
construct (not only the tag).

Good luck!

R

On 18 Jan 2014, at 17:14, Chris Fage 
mailto:cdf...@gmail.com>> wrote:

Hello Everyone,

I am currently trying to phase a structure with an asymmetric unit predicted to 
contain 20-24 monomers (space group P1). The native crystals, while beautiful 
in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and 
SeMet-derived crystals grow with poor morphology (small needles). Also, based a 
fluorescence scan, I know that mercury does not bind appreciably. Other than 
screening for a new space group, what options might I have for phasing this 
many monomers at lower resolution? Is there any real chance of solving the 
structure in this space group?

Thank you in advance for any suggestions!

Regards,
Chris


Roberto A. Steiner
Group Leader
Randall Division of Cell and Molecular Biophysics
King's College London
roberto.stei...@kcl.ac.uk

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London

Phone 0044 20 78488216
Fax0044 20 78486435

[ccp4bb]

[venkatareddy dadireddy]

Dear All,

My protein is cloned in pET-15b vector, contains His- tag, thrombin
cleavage site and extra sequence of 20 amino acids from vector. I
crystallized without cleaving extra sequence and never got any crystal
hits/crystals. Once, I tried thrombin reaction and it didn't work. Here I
would like to know how efficient the cleavage by thrombin and it is kind of
you, can provide protocol and buffer system for reaction.

Thank you
Venkat.

Re: [ccp4bb] Phasing with Many Monomers/AU

[James Holton]

The problem of many monomers in the "ASU" is not restricted to 
macromolecules. An interesting recent small molecule example is the 
structure of L-tryptophan (http://dx.doi.org/10.1107/S0108768112033484) 
which, amazingly, was not published until 2012.  This is perhaps in part 
due to difficulty in accepting 16 monomers in the ASU (they call this 
Z=16), which was unprecedented.


As a beamline scientist, I have seen "high Z" macromolecular crystals on 
many occasions, but they almost never get solved. Yes, they don't 
diffract well, but neither does anything else in the early stages of a 
project.  The reason for not solving them seems more psychological than 
anything else. The prospect of amplifying the building and refinement 
headache by a factor of "Z" when Z > 10 is perhaps too much for an early 
term graduate student to bear.


On the other hand, automated building and refinement has come a long 
way, and 24-fold NCS is a great restraint if you can get it! In fact, 
for virus structures, it has been shown that you can phase the structure 
starting with nothing but a crude spherical envelope and lots of density 
modification (http://dx.doi.org/10.1107/S0108767391013211).


 but your initial problems are going to be phasing.  Ideally what you'd 
want is a way of folding back NCS information into the heavy atom 
finding and phase refinement process, but I know of no programs that 
actually do that.  In fact, both molecular replacement and heavy-atom 
finding are hindered by this "pseodo-translation" rather than helped by 
it.  Personally, I blame the fact that methods developers seldom get 
their hands on "interesting" datasets like yours.  And if you look in 
the PDB there are very few examples of "high Z'" structures.  Ahem.


Best advice I can give is to try the "usual" approach, but look very 
seriously for NCS as early as you can.  Then apply building/phasing 
packages like shelx{cde}, phenix.autobuild, or the newly-released Crank2.


-James Holton
MAD Scientist

On 1/18/2014 11:18 PM, Felix Frolow wrote:

Francis, It can happened
We have (not yet published)  P1 with 24 molecules. When we cut His-tag we get 
P1 with 32 molecules.
In our case we believe it is dictated by very strong interaction between two 
monomers, and strong interaction between dimers with build a flattish tetramer. 
Probably such formations
is more difficult to oaks than globular oligomers.
In this moment I do not recall what we see in solution, I have to check.
Relating to structure solution, P1 is very convenient space group.
I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX or 
SHARP). For the native - molecular replacement.
In our time after tremendous developments in Refmac and Phenix and development 
o DM refinement is 3-3.4 Ang. Is not very difficult.
I would use in addition to NCS restraints in refinement also multi crystal 
averaging. Roumors say it is the most strongest phasing method (attributed to 
Eleanor Dodson, myself never used it).


FF

Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Jan 19, 2014, at 08:48 , Francis Reyes  wrote:


You sure about this space group? 24 monomers in P1 is unusual (at least to me)

F


On Jan 18, 2014, at 9:14 AM, Chris Fage  wrote:

Hello Everyone,

I am currently trying to phase a structure with an asymmetric unit predicted to 
contain 20-24 monomers (space group P1). The native crystals, while beautiful 
in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and 
SeMet-derived crystals grow with poor morphology (small needles). Also, based a 
fluorescence scan, I know that mercury does not bind appreciably. Other than 
screening for a new space group, what options might I have for phasing this 
many monomers at lower resolution? Is there any real chance of solving the 
structure in this space group?

Thank you in advance for any suggestions!

Regards,
Chris

Re: [ccp4bb] Isolation of protein-protein complexes.

[Enrico Stura]

Dear David,

Following the discussion I am starting to wonder if I have been doing  
something wrong all these years.
I always forgot to purify the complexes, I just mixed the two  
macromolecules and did the crystallization experiments.
And I will admit that I did not even worry about getting the right  
stoichiometry. It is true that I got

crystals with the wrong stoichiometry:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,  
Silverman, G.J. & Charbonnier, J.-B. (2001) Crystallization of  
macromolecular complexes: Stoichiometric variation screening. J. Cryst.  
Growth 232: 580-590.
But then ... I thought that as long as I followed the standard rules for  
crystal growth, even stoichiometry variation could be part of

standard crystallization methodology.

Since you still got all the details of the interaction from the structure  
even with extra molecules, I was not ashamed of my mistake and tried

to publish the structure. The referees did not seem to mind:
 Graille, M., Stura, E. A., Taussig, M. J., Corper, A., Sutton, B. J.,  
Charbonnier, J.-B. & Silverman, G. J. (2000) Crystal structure of a  
Staphylococcus aureus protein A domain complexed with the Fab fragment of  
a human IgM antibody: structural basis for recognition of B-cell receptors  
and superantigen activity. Proc. Natl. Acad. Sci. USA. 97: 5399-5404.


Enrico.


On Tue, 21 Jan 2014 18:02:48 +0100, Eugene Osipov   
wrote:



May be your complex components interact with column? I mean: you have
calibration plot for column, right? Does the molecular mass of proteins
calculated from elution volume equals to mass calculated by another  
method

(like SDS-PAGE)?


2014/1/21 Keller, Jacob 

 Maybe there is something required for interaction that was in the  
buffer

used for the other binding studies, but not in your SEC buffer?



JPK



*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf  
Of *David

Briggs
*Sent:* Tuesday, January 21, 2014 10:52 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Isolation of protein-protein complexes.



Dear all,



sorry for the slightly off topic post,



I have 2 proteins that have been shown to interact, by multiple groups,
and by multiple techniques - namely ELISA, SPR and DPI.



The Kd of the interaction as determined by SPR is on the order of 1 nM.



I would very much like to crystallise this protein-protein complex, and  
as
a first step I attempted to purify the complex by mixing the two  
proteins

(same protein preps and same buffers as the SPR experiment) and then
running them down a gel filtration column (Superose 6 - predicted size  
of

the complex is ~500kDa).



Somewhat irritatingly the two proteins separate beautifully on the  
column

into two distinct peaks. There is no trace of complex formation when the
peaks are analysed by SDS-PAGE.



As far as I am aware, two proteins that interact this strongly should
remain associated during gel filtration, and I was wondering if anyone  
else

has encountered anything similar in the past, and if they managed to
resolve the problem, how they went about it?



Cheers in advance,



Dave


David C. Briggs PhD
http://about.me/david_briggs








--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Isolation of protein-protein complexes.

[Eugene Osipov]

May be your complex components interact with column? I mean: you have
calibration plot for column, right? Does the molecular mass of proteins
calculated from elution volume equals to mass calculated by another method
(like SDS-PAGE)?


2014/1/21 Keller, Jacob 

>  Maybe there is something required for interaction that was in the buffer
> used for the other binding studies, but not in your SEC buffer?
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *David
> Briggs
> *Sent:* Tuesday, January 21, 2014 10:52 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Isolation of protein-protein complexes.
>
>
>
> Dear all,
>
>
>
> sorry for the slightly off topic post,
>
>
>
> I have 2 proteins that have been shown to interact, by multiple groups,
> and by multiple techniques - namely ELISA, SPR and DPI.
>
>
>
> The Kd of the interaction as determined by SPR is on the order of 1 nM.
>
>
>
> I would very much like to crystallise this protein-protein complex, and as
> a first step I attempted to purify the complex by mixing the two proteins
> (same protein preps and same buffers as the SPR experiment) and then
> running them down a gel filtration column (Superose 6 - predicted size of
> the complex is ~500kDa).
>
>
>
> Somewhat irritatingly the two proteins separate beautifully on the column
> into two distinct peaks. There is no trace of complex formation when the
> peaks are analysed by SDS-PAGE.
>
>
>
> As far as I am aware, two proteins that interact this strongly should
> remain associated during gel filtration, and I was wondering if anyone else
> has encountered anything similar in the past, and if they managed to
> resolve the problem, how they went about it?
>
>
>
> Cheers in advance,
>
>
>
> Dave
>
> 
> David C. Briggs PhD
> http://about.me/david_briggs
>



-- 
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

Re: [ccp4bb] Isolation of protein-protein complexes.

[David Briggs]

Hi all,

Thanks for the incredibly fast and helpful responses, both on and off list.

To provide some further information. I have tried the SEC with several
buffers, including buffers identical to the SPR and DPI experiments.

To-date all SEC and SPR experiments have been conducted at room temperature
- so suggestions to repeat SEC in the cold room are useful and might yield
complex.

I am also fortunate enough that only one protein is (His) tagged, so
co-purification on an IMAC resin is also something that I shall try.

Regarding the SPR, many have highlighted that the most important parameter
vis complex stability is Koff - I don't have those numbers right now (on
the train) but from memory the off rate is slow, and the interaction does
not appear to be the described fast-on/fast-off non-specific interaction.
I always run a blank for all my SPR experiments and no non-specific
interactions with the blank surface were seen

Thanks once again everyone,

Dave
On 21 Jan 2014 15:51, "David Briggs"  wrote:

> Dear all,
>
> sorry for the slightly off topic post,
>
> I have 2 proteins that have been shown to interact, by multiple groups,
> and by multiple techniques - namely ELISA, SPR and DPI.
>
> The Kd of the interaction as determined by SPR is on the order of 1 nM.
>
> I would very much like to crystallise this protein-protein complex, and as
> a first step I attempted to purify the complex by mixing the two proteins
> (same protein preps and same buffers as the SPR experiment) and then
> running them down a gel filtration column (Superose 6 - predicted size of
> the complex is ~500kDa).
>
> Somewhat irritatingly the two proteins separate beautifully on the column
> into two distinct peaks. There is no trace of complex formation when the
> peaks are analysed by SDS-PAGE.
>
> As far as I am aware, two proteins that interact this strongly should
> remain associated during gel filtration, and I was wondering if anyone else
> has encountered anything similar in the past, and if they managed to
> resolve the problem, how they went about it?
>
> Cheers in advance,
>
> Dave
> 
> David C. Briggs PhD
> http://about.me/david_briggs
>

Re: [ccp4bb] Isolation of protein-protein complexes.

[Keller, Jacob]

Maybe there is something required for interaction that was in the buffer used 
for the other binding studies, but not in your SEC buffer?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David 
Briggs
Sent: Tuesday, January 21, 2014 10:52 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Isolation of protein-protein complexes.

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple groups, and by 
multiple techniques - namely ELISA, SPR and DPI.

The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, and as a 
first step I attempted to purify the complex by mixing the two proteins (same 
protein preps and same buffers as the SPR experiment) and then running them 
down a gel filtration column (Superose 6 - predicted size of the complex is 
~500kDa).

Somewhat irritatingly the two proteins separate beautifully on the column into 
two distinct peaks. There is no trace of complex formation when the peaks are 
analysed by SDS-PAGE.

As far as I am aware, two proteins that interact this strongly should remain 
associated during gel filtration, and I was wondering if anyone else has 
encountered anything similar in the past, and if they managed to resolve the 
problem, how they went about it?

Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs

Re: [ccp4bb] Isolation of protein-protein complexes.

[Bosch, Juergen]

Hey David,

the 1 nM Kd by SPR sounds fishy to me - did you do these measurements yourself ?
Unless this is an antibody-protein complex or nanobody-protein complex or 
protein-small molecule complex, the value is too low (for a normal PPI, I would 
expect  >50 -500 nM). Possible explanation for the SPR result is non-specific 
binding of the ligand (your protein in solution) to the chip.
Which would in turn also explain your negative result in SEC eventually (what 
SEC material did you use ? Is it at the pH negatively charged perhaps - like 
the SPR chip).
Are you running the SEC in the same buffer as used in either ELISA, SPR or DPI ?
Most likely the main difference between those experiments and yours is the 
temperature, as I assume you ran your SEC at 4˚C.
Just a thought.

Jürgen


On Jan 21, 2014, at 10:51 AM, David Briggs 
mailto:drdavidcbri...@gmail.com>> wrote:

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple groups, and by 
multiple techniques - namely ELISA, SPR and DPI.

The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, and as a 
first step I attempted to purify the complex by mixing the two proteins (same 
protein preps and same buffers as the SPR experiment) and then running them 
down a gel filtration column (Superose 6 - predicted size of the complex is 
~500kDa).

Somewhat irritatingly the two proteins separate beautifully on the column into 
two distinct peaks. There is no trace of complex formation when the peaks are 
analysed by SDS-PAGE.

As far as I am aware, two proteins that interact this strongly should remain 
associated during gel filtration, and I was wondering if anyone else has 
encountered anything similar in the past, and if they managed to resolve the 
problem, how they went about it?

Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] Isolation of protein-protein complexes.

[Philippe Leone]

Dear Dave,

Indeed, an interaction in the nM range is strong, but its dynamic is 
important. If the SPR sensorgram, if published, looks like a 'crenel', 
this indicates high association/dissociation rates. In that case, your 
complex can dissociate during the GF run.
You can try crystallisation at 4°C in order to lower the dynamic of the 
interaction, this worked for me...

Good luck.

Philippe


Le 21/01/14 16:51, David Briggs a écrit :

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple 
groups, and by multiple techniques - namely ELISA, SPR and DPI.


The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, 
and as a first step I attempted to purify the complex by mixing the 
two proteins (same protein preps and same buffers as the SPR 
experiment) and then running them down a gel filtration column 
(Superose 6 - predicted size of the complex is ~500kDa).


Somewhat irritatingly the two proteins separate beautifully on the 
column into two distinct peaks. There is no trace of complex formation 
when the peaks are analysed by SDS-PAGE.


As far as I am aware, two proteins that interact this strongly should 
remain associated during gel filtration, and I was wondering if anyone 
else has encountered anything similar in the past, and if they managed 
to resolve the problem, how they went about it?


Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs



--
Philippe Leone
AFMB-UMR7257
Team 'Structural Immunology'
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33 491 82 55 93
Fax: +33 491 26 67 20
e-mail: philippe.le...@afmb.univ-mrs.fr

[ccp4bb] Isolation of protein-protein complexes.

[David Briggs]

Dear all,

sorry for the slightly off topic post,

I have 2 proteins that have been shown to interact, by multiple groups, and
by multiple techniques - namely ELISA, SPR and DPI.

The Kd of the interaction as determined by SPR is on the order of 1 nM.

I would very much like to crystallise this protein-protein complex, and as
a first step I attempted to purify the complex by mixing the two proteins
(same protein preps and same buffers as the SPR experiment) and then
running them down a gel filtration column (Superose 6 - predicted size of
the complex is ~500kDa).

Somewhat irritatingly the two proteins separate beautifully on the column
into two distinct peaks. There is no trace of complex formation when the
peaks are analysed by SDS-PAGE.

As far as I am aware, two proteins that interact this strongly should
remain associated during gel filtration, and I was wondering if anyone else
has encountered anything similar in the past, and if they managed to
resolve the problem, how they went about it?

Cheers in advance,

Dave

David C. Briggs PhD
http://about.me/david_briggs

[ccp4bb] BBSRC iCASE studentship in Leicester sponsored by MRC Technology

[Bayliss, Richard W.A. (Dr.)]

Structural and chemical biology approaches to characterize protein-protein 
interactions that regulate kinases
We are looking for a talented and highly motivated student to join us for a 
4-year BBSRC iCASE PhD studentship sponsored by MRC Technology.  The project 
will be supervised jointly by Dr. Richard Bayliss and Prof. Mark Carr in the 
Biochemistry Department at Leicester. Our groups provide an excellent 
environment for training and research in structural biology methods relevant to 
drug discovery. The project also involves an internship at MRC Technology to 
gain direct experience of drug discovery in a non-academic setting.

For further details, including how to apply, see:
http://www2.le.ac.uk/study/research/funding/kinases-regulation

The studentship is available only for UK or UK-based EU students, includes 
full-time registration and is payable as a full UK/EU tuition fee waiver and 
stipend for four years.

The successful applicant will also receive a generous financial package 
comprising a research support budget, stipend supplement, fixed expenses for 
travel and accommodation in London, and costs towards a laptop computer.


=
Dr Richard Bayliss, Reader in Structural Biology
Department of Biochemistry
Henry Wellcome Building
University of Leicester
Lancaster Road, Leicester
LE1 9HN

Tel: 0116 2297100
Web: 
http://www2.le.ac.uk/departments/biochemistry/staff/richard-bayliss/research
Twitter: @baylisslab

[ccp4bb] Job opening at Diamond Light Source, UK

[David Hall]

Hi

Diamond Light Source provides the UK and International community with world 
class tools for structural biologists and has an active collaborative community 
of crystallographers and software scientists and engineers as well as being 
co-located alongside the CCP4 core team at the Research Complex at Harwell.

We currently have a position for a software scientist or macromolecular 
crystallographer with programming experience to work with our beamline 
scientists, software development teams and users to implement new 
functionalities for MX beamlines.  Systems actively under development include 
multi-axis goniometry, multivariate X-ray focussing optics and beamline 
diagnostics which require further implementation into our software environment 
to provide for optimised macromolecular crystal data collections.

Understanding user requirements is a key element of this role to deliver the 
best solutions and to aid with this, this post will have a degree of beamline 
user support associated with it - training for this will be provided as 
required.

Details of the post and how to apply can be found here: 
http://www.diamond.ac.uk/Home/Jobs/Current/DIA0899-CH.html

Please feel free to make informal enquiries to myself but please formally apply 
via the link above.

Best wishes

Dave Hall

I04 PBS & MX Village Coordinator | Diamond Light 
Source
t: 01235 778926  |  e: david.h...@diamond.ac.uk
http://www.diamond.ac.uk/mx





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