Re: [ccp4bb] ITC question -dimer vs monomer

[DUMAS Philippe (IGBMC)]

Dear Bernhard 
I share your intuition: we should expect to observe different shapes of 
titration curves depending on whether A or B2 is in the syringe (titration and 
reverse titration). 
I suppose that you want to test your hypothesis that, eventually, you get A2B2. 
Independently of any kinetic considerations , one should consider the 
possibility that the successive equilibria A+B2<-->AB2 and A + AB2 <-->A2B2 
have distinct Kds and distinct DeltaH (which would of course be more favorable 
to detect these two distinct binding events, if they exist). 
I therefore suggest that you perform both possible titrations (if each protein 
can be sufficiently concentrated to be in the syringe) and that you try to fit 
each titration curve with the same model of interaction. If this works well, 
then the must would be to fit both titration curves at the same time with the 
same model of interaction . This is certainly the most demanding method to test 
a hypothesis and this is not at all equivalent to fit independently the two 
kinds of titration curves as you can imagine. (Let's say that this would amount 
to refine a single molecular model against crystal data from two space 
groups).The problem is of the practical possibility of doing such a joint fit 
with the available programs. I personally do such things with my own (not 
user-friendly!) programs. As far a I know, this is not possible, neither with 
Origin or PEAQ from Malvern, nor with NanoAnalyze from TA. I know that 
AFFINImeter is quite flexible to allow using specific models, but I'm not sure 
it would allow you to make such a global fit. I don't know about the 
possibility of SEDPHAT developed by P. Schuck. 
I hope this fits with your expectations. 
Best 
Philippe Dumas 



De: "Bernhard Rupp"  
À: CCP4BB@JISCMAIL.AC.UK 
Envoyé: Jeudi 3 Octobre 2019 17:05:56 
Objet: [ccp4bb] ITC question -dimer vs monomer 



Hi Fellows, 



please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer. 

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form. 



Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa. 

In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex 

is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects). 



If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s. 



Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent). 



Can someone guide me towards literature about this or perhaps share some 
first-hand experience? 



Many thanks, BR 



-- 

Bernhard Rupp 

[ http://www.hofkristallamt.org/ | http://www.hofkristallamt.org/ ] 

[ mailto:b...@hofkristallamt.org | b...@hofkristallamt.org ] 

+1 925 209 7429 

+43 676 571 0536 

-- 

Many plausible ideas vanish 

at the presence of thought 

-- 






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Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

No, the law of mass action does not depend on whether A is titrated into B or 
vice versa. The heat measured is proportional to the complex formed, relative 
to one partner being kept at constant concentration (the referece in the cell). 
The law of mass action can be re-written to describe the complex concentration 
itself as a function of A, B and Kd. Assuming a one-to-one stoichiometry, the 
function contains sums of each variable being substracted by a root function:

AB = 0.5 [ Atotal + Btotal + Kd - sqrt{  (Atotal + Btotal + Kd)^2 - 
4*Atotal*Btotal   }]

It is correct that these values should be roughly in the same range. This, 
however, is not restricted to ITC solely, but to HSQC or fluorescence-based 
titrations too. Its a direct consequence of the law of mass action.

In order to fully saturate either binding partner, say fully saturate A; AB/A 
approaching 1, all three factors need to be in the same range. The same would 
hold with B in the cell, saturating AB/B. The underlaying mass action is the 
same, just the non-ligated reference is exchanged.

For a stochiometry different from 1:1, the mass action does not yield in an 
explicit solution like the one written above. To force such a solution, one 
concentration is multiplied by a factor, N, assuming N independent, 
non-cooperative binding sites for N ligands. Lets assume a 1:2 in your case, 
2A+B2 <-> A2B2. Substituting Btotal with N*Btotal would allow to assume a 
one-to-one model with N=2. If you decide to exchange the titrant, N now 
corrects the other partner, yielding in N=0.5.





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Roger Rowlett 

Sent: Thursday, October 3, 2019 8:36:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Won't this depend on the relative final concentrations of A and B in the two 
experiments? If A going into excess B will have different mass action 
considerations that B going into excess A. Even if the final concentrations of 
A and B are stoichiometric, the initial stages of the titration will have very 
different mass action products for A into B vs. B into A. An additional wrinkle 
is the concentrations of A and B relative to the dissociation constant Kd. The 
titration curve math gets a little more complex when the concentration of the 
species is in the same order of magnitude as the Kd. There are quite a few 
examples of bollixed binding curves in the literature for tight-binding 
equilibria that ignore the relationship between Kd and ligand concentrations.  
Cooperativity issues will of course perturb any pure, non-cooperative 
statistical analysis based on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
mailto:hofkristall...@gmail.com>> wrote:
I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the 

Re: [ccp4bb] ITC question -dimer vs monomer

[Roger Rowlett]

Won't this depend on the relative final concentrations of A and B in the
two experiments? If A going into excess B will have different mass action
considerations that B going into excess A. Even if the final concentrations
of A and B are stoichiometric, the initial stages of the titration will
have very different mass action products for A into B vs. B into A. An
additional wrinkle is the concentrations of A and B relative to the
dissociation constant Kd. The titration curve math gets a little more
complex when the concentration of the species is in the same order of
magnitude as the Kd. There are quite a few examples of bollixed binding
curves in the literature for tight-binding equilibria that ignore the
relationship between Kd and ligand concentrations.  Cooperativity issues
will of course perturb any pure, non-cooperative statistical analysis based
on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
wrote:

> I am not looking for anything yet – I wonder what – if any – the
> consequences of doing it one way or the other would be.
>
> I am reasonably certain that any difference affects the analysis.
>
>
>
> Thx, BR
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Keller,
> Jacob
> *Sent:* Thursday, October 3, 2019 17:41
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] ITC question -dimer vs monomer
>
>
>
> I don’t understand what you are trying to do—are you trying to show, by
> the difference in ITC response, that the predictions you made about the
> oligomerization are true?
>
>
>
> JPK
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> 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.
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* Thursday, October 3, 2019 11:06 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] ITC question -dimer vs monomer
>
>
>
> Hi Fellows,
>
>
>
> please let me ask the respective experts an ITC question: I have 2
> proteins, stable and dialyzed in identical buffer.
>
> A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
> dimer will form.
>
>
>
> Intuitively, it should make a difference whether I titrate the dimer with
> the monomer or vice versa.
>
> In the first case, a momomer would initially meet a lot of free dimers,
> and I would expect that randomly, a AB2 complex
>
> is more likely to form than a A2B2 (let’s disregard any more complex
> colligative/cooperative effects).
>
>
>
> If I drip the dimer into the monomer pool, it is quite likely that the B
> dimer meets 2 free As, and I get right away a higher population of A2B2s.
>
>
>
> Maybe at dilutions of ITC and with sufficient equilibration that is not an
> issue at all (again, absent any cooperative effects that might alter the
> first Kd vs. the second, despite the sites on the dimer are at least
> initially equivalent).
>
>
>
> Can someone guide me towards literature about this or perhaps share some
> first-hand experience?
>
>
>
> Many thanks, BR
>
>
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
> 
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>

[ccp4bb] Position Available: Crystallography Facility Manager (Research Assistant Professor) - CUNY Advanced Science Research Center, New York City

[Keedy, Daniel]

To the ccp4bb community,

The Structural Biology Initiative ( https://asrc.gc.cuny.edu/structbio ) of the 
CUNY Advanced Science Research Center has an available position for an X-ray 
Crystallography Facility Manager (Research Assistant Professor) starting 
immediately.

The CUNY Advanced Science Research Center is a new cutting-edge research 
institution located in New York City, bringing together five Initiatives whose 
research areas span many length scales: Structural Biology, Nanoscience, 
Photonics, Neuroscience, and Environmental Science. The Structural Biology 
Initiative (SBI) features seven tenure-track and research faculty members, 
pursuing research problems across biological and chemical topics using 
techniques as diverse as X-ray diffraction, cryo-EM, NMR, solution biophysics, 
and advanced microscopy. The SBI also hosts state-of-the-art core facilities in 
three different areas: nuclear magnetic resonance (NMR) spectroscopy, mass 
spectrometry (MS), and X-ray crystallography.

The Structural Biology Initiative seeks a Facility Manager/Research Assistant 
Professor for the X-ray Macromolecular Crystallography Facility, which is 
already equipped with a Rigaku Alchemist DT liquid handler, two ARI Gryphon 
crystallization robots, and two Rigaku Minstrel DT crystal imagers at room 
temperature and 4°C. The selected candidate will be responsible not only for 
directing day-to-day operations of the facility in concert with users from the 
ASRC and externally, but also for developing an independent research program 
that leverages emerging technologies in X-ray crystallography.

The ASRC is located in upper Manhattan, immediately adjacent to the New York 
Structural Biology Center (NYSBC), a consortium of CUNY and eight other New 
York academic institutions with world-class cryo-EM, NMR, and X-ray facilities 
on-site and at Brookhaven National Laboratory. BNL, located just 70 miles from 
the ASRC, includes a newly constructed synchrotron, National Synchrotron Light 
Source II, which opened in 2015. NSLS-II is now the brightest synchrotron light 
source in the United State, offering X-rays that are 10,000x brighter than the 
original NSLS. The ASRC Structural Biology Initiative has access to several 
cutting-edge microfocus beamlines equipped for automated crystal handling and 
data collection at NSLS-II, including the NYX beamline via CUNY’s membership in 
NYSBC and the AMX and FMX beamlines via proposals.

Please find more detailed information about the position and officially apply 
via the following URL:

https://cuny.jobs/new-york-ny/macromolecular-crystallization-facility-manager-research-assistant-professor-structural-biology/E71B43986B2A4CC9920D1A83B5A90569/job/

Review of applications will begin on October 16.

Best regards,

Daniel Keedy

—
Daniel A. Keedy, Ph.D.
Assistant Professor
Structural Biology Initiative, CUNY Advanced Science Research Center
Department of Chemistry and Biochemistry, City College of New York
Biochemistry and Chemistry Ph.D. Programs, CUNY Graduate Center
www.keedylab.org   |  
dke...@gc.cuny.edu (NOT @asrc)  |  212-413-3246




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[ccp4bb] Cryo-EM Faculty Position

[Lawrence, C Martin]

Dear CCP4 Community,

Montana State University invites applications for a tenure-track faculty 
position in cryogenic electron microscopy (cryo-EM; single particle and/or 
tomography).  The position is formally a joint appointment between the 
Department of Chemistry and Biochemistry, and the Department of Microbiology 
and Immunology.  The candidate will join an active structural biology group 
with existing expertise in X-ray crystallography, NMR and mass spectrometry, 
and will include access to the University’s newly acquired Talos Arctica 
microscope equipped with a Gatan K3 camera.

Montana State University maintains a highly collaborative, interdisciplinary 
environment with research programs broadly focused in multiple areas, including 
cell and developmental biology, neurobiology/neuroscience, metalloproteins and 
metals in biology, innate and acquired immunity, virology, pathogen 
microbiology, host/pathogen interactions, microbial biofilms and environmental 
microbiology.  There are numerous opportunities for collaboration in these 
areas.

Montana State University is located in the Northern Rocky Mountain community of 
Bozeman Montana.  Bozeman is regularly recognized for its high quality of life, 
including its outdoor, cultural and educational amenities:  
http://www.montana.edu/marketing/about-msu/bozeman/.

For further details and to apply, visit:  
(https://jobs.montana.edu/postings/18200)

Thank you,
Martin

Martin Lawrence
Professor of Chemistry and Biochemistry
Montana State University
Bozeman, MT 59717
[jbcsig1.png]



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Re: [ccp4bb] ITC question -dimer vs monomer

[Rajiv gandhi.s]

Dear Dr.BR

There could be two possibilities,  you could try to do titration of dimer
into monomer loaded in cell or vice versa, and with subtraction of heat of
dilution from protein-protein titration. These kind of molecular
interaction involving distinct oligomer units need be addressed  by any
other orthogonal methods.

Regards
Rajivgandhi Sundaram


On Thu, Oct 3, 2019, 10:21 PM clare stevenson (JIC) <
clare.steven...@jic.ac.uk> wrote:

> I would try it both ways and see what you get.  Also do controls of buffer
> into each protein
>
>
>
> For extra info could also try with SPR.  Always best to do these things
> using multiple complimentary methods
>
>
>
> Best wishes
>
>
>
> Clare
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* 03 October 2019 16:06
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] ITC question -dimer vs monomer
>
>
>
> Hi Fellows,
>
>
>
> please let me ask the respective experts an ITC question: I have 2
> proteins, stable and dialyzed in identical buffer.
>
> A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
> dimer will form.
>
>
>
> Intuitively, it should make a difference whether I titrate the dimer with
> the monomer or vice versa.
>
> In the first case, a momomer would initially meet a lot of free dimers,
> and I would expect that randomly, a AB2 complex
>
> is more likely to form than a A2B2 (let’s disregard any more complex
> colligative/cooperative effects).
>
>
>
> If I drip the dimer into the monomer pool, it is quite likely that the B
> dimer meets 2 free As, and I get right away a higher population of A2B2s.
>
>
>
> Maybe at dilutions of ITC and with sufficient equilibration that is not an
> issue at all (again, absent any cooperative effects that might alter the
> first Kd vs. the second, despite the sites on the dimer are at least
> initially equivalent).
>
>
>
> Can someone guide me towards literature about this or perhaps share some
> first-hand experience?
>
>
>
> Many thanks, BR
>
>
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

As Reza already pointed out, ITC cannot tell you anything about the 
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> 
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a 
fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2.

Given the lack of additional information, its probably good to assume a simple 
one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well as 
AB2 for A), you would measure an apparent affinity and would see the 
stochiometrics according to  the inflection point (be it around equimolar 
excess or at 0.5 or 2, depending on whether you titrate A or B). If the 
reaction is more complicated and the the affinities for B2 for A differ 
significantly much from the affinity of AB2 for A, then a simple one-to-one 
would leave some notable information in the residual standard deviations 
(meaning, the residuals would not spread normally around the Regression line, 
but should show a wavy pattern).

Sorry for the long mail..

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Michael Fairhead 

Sent: Thursday, October 3, 2019 5:59:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Hello,
If you Google Alan cooper ITC insulin, you should find his work describing the 
study of insulin dimer:monomer equilibrium and the effect of cyclodextrin 
studied via ITC. This may be of some help.
Cheers
Mike

https://www.google.com/url?sa=t=web=j=http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf=2ahUKEwiOpoeisYDlAhUDThUIHX5mAyMQFjAAegQIAhAB=AOvVaw0Ng-W03upy4DG12unFGDY3


From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536

Re: [ccp4bb] ITC question -dimer vs monomer

[clare stevenson (JIC)]

I would try it both ways and see what you get.  Also do controls of buffer into 
each protein

For extra info could also try with SPR.  Always best to do these things using 
multiple complimentary methods

Best wishes

Clare

From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] ITC question -dimer vs monomer

[Michael Fairhead]

Hello,
If you Google Alan cooper ITC insulin, you should find his work describing the 
study of insulin dimer:monomer equilibrium and the effect of cyclodextrin 
studied via ITC. This may be of some help.
Cheers
Mike

https://www.google.com/url?sa=t=web=j=http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf=2ahUKEwiOpoeisYDlAhUDThUIHX5mAyMQFjAAegQIAhAB=AOvVaw0Ng-W03upy4DG12unFGDY3


From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet - I wonder what - if any - the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

[Reza Khayat]

?Isn't the entire idea of using ITC that you are measuring an equilibrium 
constant? Wouldn't this eliminate the nuances of what you're looking for (i.e. 
kinetics)? Perhaps you should use SPR to tease out this model?


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Thursday, October 3, 2019 11:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet - I wonder what - if any - the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] ITC question -dimer vs monomer

[Bernhard Rupp]

I am not looking for anything yet - I wonder what - if any - the
consequences of doing it one way or the other would be.

I am reasonably certain that any difference affects the analysis.

 

Thx, BR

 

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

 

I don't understand what you are trying to do-are you trying to show, by the
difference in ITC response, that the predictions you made about the
oligomerization are true?

 

JPK

 

+

Jacob Pearson Keller

Research Scientist / Looger Lab

HHMI Janelia Research Campus

19700 Helix Dr, Ashburn, VA 20147

Desk: (571)209-4000 x3159

Cell: (301)592-7004

+

 

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.

 

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK> > On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK  
Subject: [ccp4bb] ITC question -dimer vs monomer

 

Hi Fellows,

 

please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
dimer will form.

 

Intuitively, it should make a difference whether I titrate the dimer with
the monomer or vice versa.

In the first case, a momomer would initially meet a lot of free dimers, and
I would expect that randomly, a AB2 complex

is more likely to form than a A2B2 (let's disregard any more complex
colligative/cooperative effects).

 

If I drip the dimer into the monomer pool, it is quite likely that the B
dimer meets 2 free As, and I get right away a higher population of A2B2s.

 

Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the
first Kd vs. the second, despite the sites on the dimer are at least
initially equivalent).

 

Can someone guide me towards literature about this or perhaps share some
first-hand experience?

 

Many thanks, BR

 

--

Bernhard Rupp

http://www.hofkristallamt.org/
 

b...@hofkristallamt.org  

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

--

 

 

  _  

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Re: [ccp4bb] ITC question -dimer vs monomer

[Keller, Jacob]

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

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.

From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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[ccp4bb] ITC question -dimer vs monomer

[Bernhard Rupp]

Hi Fellows,

 

please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
dimer will form.

 

Intuitively, it should make a difference whether I titrate the dimer with
the monomer or vice versa.

In the first case, a momomer would initially meet a lot of free dimers, and
I would expect that randomly, a AB2 complex

is more likely to form than a A2B2 (let's disregard any more complex
colligative/cooperative effects).

 

If I drip the dimer into the monomer pool, it is quite likely that the B
dimer meets 2 free As, and I get right away a higher population of A2B2s.

 

Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the
first Kd vs. the second, despite the sites on the dimer are at least
initially equivalent).

 

Can someone guide me towards literature about this or perhaps share some
first-hand experience?

 

Many thanks, BR

 

--

Bernhard Rupp

http://www.hofkristallamt.org/

b...@hofkristallamt.org  

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

--

 




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https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Figure of merit in refinement

[Alexandre Ourjoumtsev]

Thank you, Eleanor, for an important reminder : 

obviously, one more recent and relevant paper is that by Read and McCoy (Acta 
Cryst, D, 2016) 

[ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784668/ | 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784668/ ] 

With best wishes, 

Sacha 

- Le 3 Oct 19, à 12:07, Eleanor Dodson  a écrit 
: 

> The maths for estimating the FOM during refinement in REFMAC is given in some
> detail in the original paper.

> However the assessment uses estimates of the observation standard uncertainly,
> and SigmaA - the estimate of the resolution dependent error due to coordinate
> errors and missing atoms -
> and both these terms can be inaccurate.

> Randy Read et al has suggested ways of improving the SigmaA estimates, and
> better data processing SHOULD help with the measurement errors..

> So - beware but that is amn outline of the teheory
> Eleanor

> Refinement of macromolecular structures by the Maximum likelihood method.
> G.N.Murshudov, A.A.Vagin, E.J.Dodson,(1997) Acta crystallogr. D53, 240-255



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Re: [ccp4bb] Figure of merit in refinement

[Eleanor Dodson]

  The maths for estimating the FOM during refinement in REFMAC is
given in some detail in the original paper.

However the assessment uses estimates of the observation standard
uncertainly, and SigmaA - the estimate of the resolution dependent
error due to coordinate errors and missing atoms -
and both these terms can be inaccurate.

Randy Read et al has suggested ways of improving the SigmaA estimates,
and better data processing SHOULD help with the measurement errors..


So - beware but that is amn outline of the teheory

Eleanor



Refinement of macromolecular structures by the Maximum likelihood method.
  G.N.Murshudov, A.A.Vagin, E.J.Dodson,(1997) Acta crystallogr. D53, 240-255


On Thu, 3 Oct 2019 at 08:45, Alexandre Ourjoumtsev <
alexander.ourjoumt...@univ-lorraine.fr> wrote:

> Dear Andre,
>
>
> I would strongly advice you to look at the article by Lunin and Skovoroda
> (Acta Cryst, A, 1995) that addresses exactly your question:
>
> https://scripts.iucr.org/cgi-bin/paper?vs0124
>
> The authors remind a very important point that after model refinement ML
> phase errors are strongly underestimated if using all reflections, as that
> was done in the original works (see references in the article). While the
> same ML estimates work perfectly for unrefined models, that's not the case
> for refined ones, as was observed yet in the beginning of 80ths.
>These authors show then that using the test-set of reflections (the
> same as for R-free) is crucial to get the correct phase error estimates and
> respective FOMs for all cases, as this is implemented now in modern
> refinement programs. See also the article by Pannu & Read (Acta Cryst, A,
> 1996)
>
> https://onlinelibrary.wiley.com/doi/pdf/10.1107/S0108767396004370
>
>
> A more recent important article on this topic is that by Praznikar and
> Turk (Acta Cryst, D, 2009)
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257616/
>
> who discuss what can be done if a statistically significant test set of
> reflections is not available.
>
> I hope this helps you.
>
>
> With best wishes,
>
> Sacha Urzhumtsev
>
>
>
> - Le 3 Oct 19, à 2:17, Andre LB Ambrosio  a écrit :
>
> Dear Jonathan, many thanks for this. I will have a look at it right away.
> With best wishes,
> Andre.
>
> On Wed, Oct 2, 2019, 7:51 PM Jonathan Cooper  wrote:
>
>> This is a very good place to start:
>>
>> https://www-structmed.cimr.cam.ac.uk/Course/Statistics/statistics.html
>>
>> Also recommend this one:
>>
>> https://doi.org/10.1107/S0108767386099622
>>
>> and Main, P. (1979) Acta Cryst. A35, 779-85 - the maths in this one are a
>> bit easier!
>>
>>
>>
>> On Wednesday, 2 October 2019, 22:47:56 BST, Andre LB Ambrosio <
>> an...@ifsc.usp.br> wrote:
>>
>>
>> Dear all,
>>
>> How is the phase error estimated for any given reflection, specifically
>> in the context of model refinement? In terms of math I mean.
>>
>> How useful is FOM in assessing the phase quality, when not for initial
>> experimental phases?
>>
>> Many thank in advance,
>>
>> Andre.
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>
> --
>
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>
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[ccp4bb] Research Associate in CryoEM/Protein Structural Biology

[Michael Lockhart]

An excellent opportunity between the University of Manchester and University of 
Leeds.


Our Faculty’s integrated structure, and state-of-the-art research facilities, 
enable a truly translational approach to biology, medicine and health, and 
provide both staff and students with a unique opportunity to have a very real 
and positive impact on people’s lives.


A postdoctoral vacancy is available within an active and well-funded research 
team to investigate the structure and function of multi-molecular complexes 
responsible for extracellular matrix organisation in the context of 
inflammatory processes and reproductive biology.


The project will use cryo-electron microscopy (cryoEM), in combination with 
other quantitative biophysical techniques, to determine the high-resolution 
structures of protein complexes that act as 'crosslinking nodes', which 
stabilise matrices containing the simple polysaccharide hyaluronan. These 
complexes are essential for ovulation and fertilisation in mammals and also 
form wherever there is inflammation, e.g. in articular joints during arthritis. 
Detailed structural insights will enable the targeted modulation of hyaluronan 
cross-linking, to interfere with pathological matrix development and to 
generate matrices with tailored properties in vitro, e.g. for applications in 
regenerative medicine and human fertility treatments.


We are seeking a post-doctoral researcher experienced in protein structure 
determination, ideally using single-particle cryoEM analysis. Knowledge of 
recombinant protein production, other structural techniques (e.g. Small-Angle 
X-ray Scattering (SAXS) and X-ray crystallography) and biophysical analysis of 
protein-ligand interactions are also desirable.


This is a full-time position, funded by the BBSRC, and is available from 1st 
December 2019, or as soon as possible thereafter, for up to three years.


The School is strongly committed to promoting equality and diversity, including 
the Athena SWAN charter for gender equality in higher education. The School 
holds a Silver Award which recognises their good practice in relation to 
gender; including flexible working arrangements, family-friendly policies, and 
support to allow staff achieve a good work-life balance. We particularly 
welcome applications from women for this post. All appointment will be made on 
merit. For further information, please visit: 
https://www.bmh.manchester.ac.uk/about/equality/

Please note that we are unable to respond to enquiries, accept CVs or 
applications from Recruitment Agencies.



Enquiries about the vacancy, shortlisting and interviews:
Name: Professor Anthony J Day.
Email: anthony@manchester.ac.uk 
Tel: 0161 2751495
Technical support:
Email: 
universityofmanches...@helpmeapply.co.uk
Tel: 0161 850 2004
General enquiries:
Email: hrservi...@manchester.ac.uk
Tel: 0161 275 4499

This vacancy will close for applications at midnight on the closing date.
Further Particulars


Job Reference : BM
Location : Oxford Road, Manchester
Closing Date (DD-MM-) : 04/11/2019
Salary : £32,236 to £35,211 per annum depending on relevant experience
Employment Type : Fixed Term
Faculty / Organisational Unit : Biology, Medicine & Health
Division : Cell Matrix Biology and Regenerative Medicine
Hours Per week : Full Time
Contract Duration : Starting from 1 December for 36 months



Kind regards
Mike

Research Associate | Baldock Lab | Division of Cell Matrix Biology and 
Regenerative Medicine | School of Biological Sciences | Faculty of Biology, 
Medical and Health Sciences | University of Manchester | C.3258 Michael Smith 
Building | Oxford Road | Manchester | M13 9PT | Tel:  (+44) 16127(51478)





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[ccp4bb] Bursaries available for CCP4 Study Weekend 2020

[Karen McIntyre - UKRI STFC]

Standard Student Bursaries

As in year's past, CCP4 has made funding available for students, to help with 
the costs of attending Study Weekend, in the form of a bursary. All students 
are eligible for a standard bursary which covers registration plus one nights 
accommodation in halls. Bursaries will be given out to all students registering 
during the early bird registration period - 2nd September to 17th November 2019.

Students obtaining a standard bursary will only need to pay for any additional 
night's accommodation.


Travel Bursaries

Limited funds will also be made available to help pay travel costs for students 
and young post-docs from non-UK laboratories. Please send a separate letter, 
signed by your supervisor and stating the expected cost of travel in pounds 
sterling, to justify applications for travel support. Supervisors are asked to 
nominate only one candidate from their Laboratory. Delegates are expected to 
travel by the most economic means possible. Candidates from less favoured 
regions will be given preference.

Applications should be sent to:

Georgia Lomas
CCP4 Study Weekend Travel Bursary
Science and Technology Facilities Council
Rutherford Appleton Laboratory
RCaH 1.22
Harwell Oxford
Didcot
OX11 0FA, UK

You can also fax copies of your letters to Georgia on +44 (0)1235 567720 or 
e-mail ccp...@stfc.ac.uk.

Applications for travel bursaries need to be in by Thursday 31st October 2019.

Regards

Karen McIntyre
Science & Technology Facilties Council
Scientific Computing Department - CCP4
RCaH 1.22

Tel +44 (0) 1235 44 5790
Fax  +44 (0) 1235 56 7720

[*] @ccp4_mx

STFC is part of UK Research and Innovation
For more information visit https://stfc.ukri.org/

[cid:image002.png@01D4C399.5E033C70]


**Please note that I only work mornings until 1.30pm**

From: CCP4 bulletin board  On Behalf Of Jon Agirre
Sent: 11 September 2019 10:16
To: ccp4bb 
Subject: [ccp4bb] CCP4 Study Weekend 2020: registration now open!

Dear colleagues,

We are pleased to announce that the next CCP4 Study Weekend in its 2020 edition 
will be devoted to the single most synergic task in structural biology: model 
building. Aside from covering the latest advances in both automated and manual 
procedures, the meeting will offer sessions on related topics such as 
refinement, validation and representation, with a focus on helping you produce 
the best possible model from your experimental data. All sessions will have an 
integrative character, presenting methods and approaches that may be 
indistinctly used with X-ray crystallography, and electron cryo-microscopy or 
diffraction.

This meeting will have a couple of innovations too - more announcements will 
follow in due course. The most important ones will be posted here (as always), 
but for more fluid news, speaker profiles and direct communication with the 
organisers, please use hashtag #CCP4SW on Twitter or give us a follow (vide 
infra).

As in recent editions, the event will be held at the East Midlands Conference 
Centre in Nottingham, from the 7th to the 9th of January 2020. We are very much 
looking forward to welcoming you in Nottingham. And yes, there will be a 
Cèilidh 
(https://en.wikipedia.org/wiki/Cèilidh).

Registration and programme details: https://tinyurl.com/ccp4sw2020

Best wishes,

Robbie Joosten (@Robbie_Joosten),
Alan Roseman (@AlanRoseman3),
Jon Agirre (@glycojones),
and the rest of CCP4 (@ccp4_mx)
--
Dr Jon Agirre
Royal Society University Research Fellow
York Structural Biology Laboratory / Department of Chemistry
University of York, Heslington, YO10 5DD, York, UK
http://www.york.ac.uk/chemistry/research/ysbl/people/staff/jagirre/
Office: /B/K/065 Phone: +44 (0) 1904 32 8252
Twitter: @glycojones



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Re: [ccp4bb] Figure of merit in refinement

[Alexandre Ourjoumtsev]

Dear Andre, 

I would strongly advice you to look at the article by Lunin and Skovoroda (Acta 
Cryst, A, 1995) that addresses exactly your question: 

[ https://scripts.iucr.org/cgi-bin/paper?vs0124 | 
https://scripts.iucr.org/cgi-bin/paper?vs0124 ] 

The authors remind a very important point that after model refinement ML phase 
errors are strongly underestimated if using all reflections, as that was done 
in the original works (see references in the article). While the same ML 
estimates work perfectly for unrefined models, that's not the case for refined 
ones, as was observed yet in the beginning of 80ths. 
These authors show then that using the test-set of reflections (the same as for 
R-free) is crucial to get the correct phase error estimates and respective FOMs 
for all cases, as this is implemented now in modern refinement programs. See 
also the article by Pannu & Read (Acta Cryst, A, 1996) 

[ https://onlinelibrary.wiley.com/doi/pdf/10.1107/S0108767396004370 | 
https://onlinelibrary.wiley.com/doi/pdf/10.1107/S0108767396004370 ] 

A more recent important article on this topic is that by Praznikar and Turk 
(Acta Cryst, D, 2009) 

[ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257616/ | 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257616/ ] 

who discuss what can be done if a statistically significant test set of 
reflections is not available. 

I hope this helps you. 

With best wishes, 

Sacha Urzhumtsev 

- Le 3 Oct 19, à 2:17, Andre LB Ambrosio  a écrit : 

> Dear Jonathan, many thanks for this. I will have a look at it right away.
> With best wishes,
> Andre.

> On Wed, Oct 2, 2019, 7:51 PM Jonathan Cooper < [ mailto:bogba...@yahoo.co.uk |
> bogba...@yahoo.co.uk ] > wrote:

>> This is a very good place to start:

>> [ https://www-structmed.cimr.cam.ac.uk/Course/Statistics/statistics.html |
>> https://www-structmed.cimr.cam.ac.uk/Course/Statistics/statistics.html ]

>> Also recommend this one:

>> [ https://doi.org/10.1107/S0108767386099622 |
>> https://doi.org/10.1107/S0108767386099622 ]

>> and Main, P. (1979) Acta Cryst. A35, 779-85 - the maths in this one are a bit
>> easier!

>> On Wednesday, 2 October 2019, 22:47:56 BST, Andre LB Ambrosio < [
>> mailto:an...@ifsc.usp.br | an...@ifsc.usp.br ] > wrote:

>> Dear all,

>> How is the phase error estimated for any given reflection, specifically in 
>> the
>> context of model refinement? In terms of math I mean.

>> How useful is FOM in assessing the phase quality, when not for initial
>> experimental phases?

>> Many thank in advance,

>> Andre.

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