Re: [ccp4bb] what is isomorphous?

[Carlos Kikuti]

Hello!

I have to admit my maths is a bit lazy, but this discussion got me stitched
up, because of a point I believe has not been addressed: the Rfree flags.
I've been trained to import Rfree flags whenever the crystals have the same
space group and similar cell dimensions to the search model for molecular
replacement - this to avoid "cheating" the Rfree validation with
reflections that the search model has already 'seen'. We often work with
series of crystals of the same proteins with different ligands, which give
groups of very similar unit cells. So far my strategy has been to mirror
the Rfree flags (using the -ref or -rfree keyword in Autoproc) whenever the
biggest difference in one of the dimensions is 5% - number just out of my
instinct, taking in account the Rmerge of ~0.1 or less in the good cases.
Maximum resolutions are between 2.7 and 1.9 angstroms. Now considering the
fact that isomorphism depends on resolution, it makes me reconsider the 5%
cut-off: this might be fine at the low resolution, but what about the
higher resolution shells? What would be the best way to proceed in these
cases, then? Because the level of 'cheating' will also vary with
resolution...

Carlos

On Sun, Dec 31, 2023 at 5:21 PM James Holton  wrote:

> Ahh yes. I still have the very helpful email I got from Dame Louise
> Johnson in 2010.  I don't think she would mind my quoting it here:
>
> Dear James
>
> I was sorry to miss you when you were at Diamond - I was in Germany.
>
> The story of the two forms of lysozyme crystals goes back to about 1964 when
> it was found that the diffraction patterns from different crystals could be
> placed in one of two classes depending on their intensities.  This discovery
> was a big set back at the time and I can remember a lecture title being
> changed from the 'The structure of lysozyme' to 'The structure of lysozyme
> two steps forward and one step back'.  Thereafter the  crystals were
> screened based on intensities of  the (11,11,l) rows to distinguish them
> (e.g. 11,11,4 > 11,11,5 in one form and vice versa in another). Data were
> collected only for those that fulfilled the Type II criteria. (These
> reflections were easy to measure on the linear diffractometer because
> crystals were mounted to rotate about the diagonal axis). As I recall both
> Type I and Type II could be found in the same crystallisation batch .
> Although sometimes the external morphology allowed recognition this was not
> infallible.
>
> The structure was based on Type II crystals. Later a graduate student Helen
> Handoll examined Type I. The work, which was in the early days and before
> refinement programmes, seemed to suggest that the differences lay in the
> arrangement of water or chloride molecules (Lysozyme was crystallised from
> NaCl). But the work was never written up.  Keith Wilson at one stage was
> following this up as lysozyme was being used to test data collection
> strategies but I do not know the outcome.
>
> An account of this is given in International Table Volume F (Rossmann and
> Arnold edited 2001) p760.
>
> Tony North was much involved in sorting this out and if you wanted more info
> he would be the person to contact.
>
> I hope this is helpful. Do let me know if you need more.
>
> Best wishes
>
> Louise
>
> Armed with this advice, I searched the PDB using what I call the "Johnson
> ratio" of F(11,11,4) / F(11,11,5) and found there was a continuous spectrum
> (pasted below). The extrema of this spectrum were 3aw6 and 3aw7 (circled),
> which are not only from the same paper, but from the same crystal: a
> dehydration study.  Despite a modest unit cell size change of 0.7%, the
> R-factor between the Fobs of these two entries (aka R-iso) is 44%. Its like
> they are different proteins, and a 12% change in relative humidity was all
> it took.  I never did get a chance to tell Louise that it was a dehydration
> effect. It took me too long to figure it out. But, I expect she would have
> found that information delightful.
>
> To weigh in on the OP:
> First: @Doeke, no I am not reviewing your new paper, but I hope whomever
> is is being helpful.
>
> Second: I am with Randy Read that isomorphism means "same shape", and also
> with Bernhard Rupp that "same" is resolution dependent.  Anything is
> "isomorphous" if you stand far enough away from it (like Carl Sagan's "pale
> blue dot").  So, I personally define "isomorphism" in terms of the
> agreement between the structure factors (Fobs). When does it become
> non-isomorphism? I say this is when the changes in Fobs become intolerable.
> What is intolerable? Depends on what you are doing, but in general it is
> good to compare the effect of interest to the existing noise. If the
> changes in Fobs due to the structural shift become larger than SIGFobs,
> then you start having "non-isomorphism".  For the common example of merging
> data from multiple crystals, non-isomorphism becomes intolerable when it is
> large enough to degrade rather than 

[ccp4bb] Post-doc in Paris

[Carlos Kikuti]

The Structural Motility team, directed by Anne Houdusse at the Curie
Institute (Paris Center), is searching for a post-doctoral fellow.

This dynamic laboratory is looking for an expert in sample preparation and
structural determination via X-ray crystallography or cryoEM studies. We
use a multi-disciplinary approach combining structural biology, chemical
biology and cell biology to innovate in the investigation and understanding
of molecular mechanisms involving different motors in cells. Our studies
concern the very basics of force production, as well as the different
cellular roles of the motors, their regulation, and how they can be
targeted for developing novel chemical biology tools and therapies against
human diseases.
 We are looking to recruit and train a dynamic and motivated candidate who
is eager to gain independence and take the lead on one of our exciting
scientific projects.
Experience in X-ray crystallography, including data processing, is
essential. Experience in sample preparation for cryoEM, molecular dynamics
or cell biology is a plus.
 If you are interested, please send a CV and a statement of research
interests and goals as well as three letters of recommendation from your
previous employers.
Contact : Anne Houdusse (anne.houdu...@curie.fr)
Website : Structural Motility Team https://institut-curie.org/team/houdusse



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[ccp4bb] Job opening for a postdoctoral position to join the Structural Motility team at the Curie Institute Paris, France.

[Carlos Kikuti]

*Job opening for a postdoctoral position to join the Structural Motility
team at the Curie Institute Paris, France. *



The *Structural Motility *team is searching for *a post-doctoral fellow to
join a dynamic laboratory *directed by Anne Houdusse at the Curie Institute
(Paris Center).

We are looking for an *expert in structural determination via X-ray
crystallography or cryoEM studies.  *The project aims to gain insights on
molecular motors, in particular for how their cellular role is defined and
how they can be targeted in the context of novel therapies against human
diseases.  The position will also lead to the development of *chemical
biology tools and drug candidates. *Our team indeed innovate to investigate
and understand molecular mechanisms in cells.



By joining our team, the post-doctoral fellow will be given opportunities
to direct research for challenging and important biological questions
requiring cryoEM approaches and will increase his/her knowledge on the
cytoskeletal by functional and cellular studies.  He or she will have
access to all the equipment and software necessary for solving X-ray and
CryoEM structures, including a brand new 200 kV Glacios electron microscope
equipped with a Falcon 4 camera at the Curie Institute.



*Our team studies the mechanism and function of force production by
molecular motors *and has made important discoveries on how force
generation can power cellular processes in human health and how mutations
in their function lead to disease.  A multi-disciplinary approach that
combines structural biology (x-ray and cryoEM), chemical biology and cell
biology is being used in our studies.  With such an approach, we decipher
how molecular motors produce force by visualizing yet unknown structural
states, or by understanding how their activity is regulated. We use
chemical biology and cell biology approaches to test insights from
structures.  We also investigate and develop specific modulators of the
force generated by these motors (including compounds in phase 3 clinical
trials) to guide the development of new therapies.



We are looking to recruit and train a dynamic and motivated candidate who
is eager to gain independence and take the lead on one of our exciting
scientific projects.  Experience in molecular dynamics or cell biology is a
plus.



If you are interested, please send a CV and a statement of research
interests and goals as well as three letters of recommendation from your
previous employers.

*Contact *: Anne Houdusse (anne.houdu...@curie.fr)

*Website* : Structural Motility Team
https://institut-curie.org/team/houdusse



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Re: [ccp4bb] ARP/WARP won't install - old glibc trouble

[Carlos Kikuti]

Dear Art,

I am the sysadmin, which is bad news since I am much better as a
crystallographer... and I was working on a different issue on the same
machine the past week.

Our institut has a good firewall so I believe we'll be fine, the
installation of Arp/warp was smooth after the modification of grub as you
proposed.

Thank you very much!

Carlos

On Fri, Sep 4, 2020 at 9:30 PM Artem Lyubimov  wrote:

> Hi Carlos,
>
> We had the same problem trying to install ARP/wARP on our Debian 10
> machines. TL;DR: you (or your sysadmin, since this requires root access)
> have to enable vsyscall as follows:
>
> echo 'GRUB_CMDLINE_LINUX_DEFAULT="vsyscall=emulate"' >> /etc/default/grub
> update-grub
> reboot
>
> An explainer (mostly from our sysadmin):
>
> vsyscall is a mechanism that accelerates certain system calls in Linux,
> apparently added as a way to execute specific system calls that don't need
> any real level of privilege to run. It's currently considered not very
> secure, which is why it's deactivated in many recent Linux flavors,
> including apparently Debian 10. The vsyscall = emulate setting seems to
> be the safest way to have vsyscall enabled while minimizing security
> vulnerability. Still, it seems like the best way to move forward is to
> compile the next version of ARP/wARP with the newer version of glibc. (some
> key insights obtained from this GitHub post and ones around it:
> https://github.com/microsoft/WSL/issues/1462#issuecomment-274398213
>
> Hope this helps!
> Art
>
>
>> On Thu, Sep 3, 2020 at 8:34 AM Carlos Kikuti  wrote:
>>
>>> Dear CCP4BB,
>>>
>>> I am unable to install ARP/WARP on a server with Debian 10, it says:
>>>
>>> Segmentation fault
>>> *** ERROR ***
>>> This machine cannot run ARP/wARP executables that
>>> are statically linked to glibc.
>>>
>>> *** INSTALLATION OF ARP/wARP 8.0 FAILED ***
>>> Modified version exit status 1
>>>
>>> -- after some digging I found several old, unsolved issues about that in
>>> GitHub, any ideas about how to get around?
>>>
>>>
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>>
>>
>
> --
> Artem Y. Lyubimov, PhD
> Staff Scientist
> SSRL SMB Crystallography
> 2525 Sand Hill Road mail stop 99
> Menlo Park, CA 94025-7015
> (415) 325-2057
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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[ccp4bb] ARP/WARP won't install - old glibc trouble

[Carlos Kikuti]

Dear CCP4BB,

I am unable to install ARP/WARP on a server with Debian 10, it says:

Segmentation fault
*** ERROR ***
This machine cannot run ARP/wARP executables that
are statically linked to glibc.

*** INSTALLATION OF ARP/wARP 8.0 FAILED ***
Modified version exit status 1

-- after some digging I found several old, unsolved issues about that in
GitHub, any ideas about how to get around?



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Re: [ccp4bb] size exclusion columns

[Carlos Kikuti]

I like the Xbridge columns from Waters, but for SEC-MALS you can also use
Superose 6 Increase (GE), which will take longer to equilibrate and will go
a bit noisier than a silica-based column, but might be advantageous in case
a number of your proteins decide to interact with the silica resin.

Concerning the pH, you can use an Xbridge (modified silica) up to pH 8.0 or
even pH 8.5 but keep in mind you are reducing the life of the column by
doing that.

Don't forget to ask the people who sold you the MALS detector, the serious
companies always have someone to give good advice.


On Thu, Apr 26, 2018 at 6:03 PM, Markus Heckmann 
wrote:

> Dear all,
>
> We are looking for a size exclusion chromatography column
> (silica-based) for protein purification prior to a MALS-detector. We
> looked for (www.waters.com BEH-450), Sepax (Unix-C 300) and Phenomex
> (BioZen SEC-3).  Any 'column' tips or recommendations when dealing
> with large proteins (MDa)?
>
> Many thanks
> Markus
>

Re: [ccp4bb] off topic: Alternatives to GE HiTrap columns

[Carlos Kikuti]

Well GE sells empty columns (XK for low back-pressure, tricorn for higher
back-pressure) that you can fill with the resin you want. They seem
expensive but for that kind of things I don't think any option will be real
'savings'. For nickel affinity we have been having good results with
Roche's Complete - it requires less imidazole for binding and elution, and
at the same time it is compatible with reducing agents and chelators. And
we still buy the HisTrap FF crude.

On Wed, Feb 15, 2017 at 6:03 PM, Christian Roth 
wrote:

> Hi Markus,
>
> BioRad has columns as well (prepacked and empty), which follow the
> establiched connector standard. So usable without the need of adapters.
>
> Cheers
>
> Christian
>
> Am 15.02.2017 um 16:57 schrieb Markus Seeliger:
>
> Dear all,
> we are happy users of all that GE offers around their FPLC system, but I
> am getting a little tired of feeling monopolized. Are any of you aware of
> either empty columns or prepacked columns (e.g. metal affinity or ion
> exchange resins) from other companies?
> Thanks for your advice
>
> Markus
>
>
>

Re: [ccp4bb] The binding between disordered and ordered proteins

[Carlos Kikuti]

Hmmm… is it really (physiological-like) binding, or your protein A is 
aggregating/precipitating on the partner? Do you have a good negative control 
(a similar protein to which protein A should not bind)? Also, as a general 
rule, be careful about your detection method for the pull-down, don't get too 
sensitive or you'll start seeing hair on eggs -- maybe that's not the case, 
since you say strong interaction.

Carlos


 
Em 22 oct. 2013, às 04:39, Clement Angkawidjaja 
clem...@bio.mls.eng.osaka-u.ac.jp escreveu:

 Dear Dee,
 
 Some proteins with chaperone-like activity (perhaps your B?) can only bind to 
 partially folded proteins.
 Probably A folds to a molten globule structure after 1-2 days. You can check 
 by spectroscopic techniques (ANS or Trp fluorescence, CD).  
 Hope that helps.
 
 Cheers,
 Clement
 
 On 10/22/13 11:10 AM, Xiaodi Yu wrote:
 Dear All:
 
 I have a general question about protein- protein interactions. I have two 
 proteins, A and B. A is a disordered protein while B is a well folded 
 protein. The binding between A and B has been approved by GST-pull down 
 assay previously. The strange thing is I cannot get them bind if protein A 
 were just freshly prepared. However, if I kept these two proteins separately 
 for one or two days at 4 degree and then did the GST-pull down assay again, 
 I can observe very strong interaction between A and B. 
 
 Protein A doesn't contain any cys residue. I have already test certain 
 chemicals which might affect the interactions, for example, DTT and EDTA. 
 These chemicals seems to have no effect on the binding. 
 
 Although A is a disordered protein, does it need such long time to find its 
 proper conformation?
 
 Do any people have similar experience? Any suggestions are greatly 
 appreciated.
 
 Thanks,
 
 Dee
 

[ccp4bb] Double screen bug in Xquartz, Mac OS 10.9 (Mavericks)

[Carlos Kikuti]

This is just to inform that if you have a second screen plugged to your Mac and 
you want to use Mac OS 10.9, you should consider disabling the “Displays have 
separate Spaces” option, or you won’t be able to work with Coot (the window 
will keep hiding from you). This is not a Coot bug (and probably will appear 
with other Xquartz-based software, too) it’s due to a compatibility issue 
between Xquartz and OS 10.9. More details below:

Extracted from http://xquartz.macosforge.org/trac/ticket/797 :

Came across this from the Ars Technica review of Mavericks:

Well, perhaps not all of us. If you prefer the old behavior, uncheck the 
Displays have separate Spaces checkbox in the Mission Control preference 
pane. Doing so will also restore the ability to have windows that span more 
than one display.'

This should return your desktop to the way you expect it to behave.

​http://arstechnica.com/apple/2013/10/os-x-10-9/11/

Modificado 7 horas atrás por jeremyhu@…

Yes, that is a valid workaround, but the actual fix is something that requires 
a change in OS X

“

Carlos


Em 24 oct. 2013, à(s) 00:21, Francis Reyes francis.re...@colorado.edu 
escreveu:

 I'm currently using CCP4/COOT (from the official installer) and autoPROC  on 
 Mavericks without any problems. I imagine the rest of the global phasing 
 tools will work nicely. 
 
 
 I had issues with fink, you have to use a branch from github as well as 
 manually install and update the Command Line Tools just to get fink working...
 
 
 F
 
 -
 Francis E. Reyes PhD
 215 UCB
 University of Colorado at Boulder
 On Oct 23, 2013, at 3:16 PM, Carlos Kikuti kik...@gmail.com wrote:
 
 Hi,
 
 Can I profit from Kristin's question and add one: is it too soon to know if 
 the crystallography software (CCP4, Coot, Autobuster, XDS) will work fine 
 with Mavericks (Mac OS X 10.9)? 
 
 (I remember a bit of trouble when Lion came off).
 
 Carlos
 
 Em 23 oct. 2013, às 22:10, Kristin Low kristin@queensu.ca escreveu:
 
 Hi everyone,
 
 Sorry for being a bit off topic, but I thought this group would be great 
 for advice.
 
 I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn 
 as to whether I need integrated vs discrete graphics for structural 
 biology, including molecular modelling, especially since the latest 
 advances by Intel in terms of integrated graphics. Right now with the new 
 releases, the options are between Intel Iris Pro (5200 series) and Intel 
 Iris Pro + Nvidia GT 750M. Thoughts?
 
 
 Thanks everyone!
 
 Kristin
 
 
 -
 Kristin Low
 Ph.D. Candidate
 Queen's University
 Department of Biomedical and Molecular Sciences
 Botterell Hall, Room 645
 Kingston, ON
 Canada  K7L 3N6
 
 tel: +1-613-533-3019
 -
 
 
 
 
 
 
 
 
 

Re: [ccp4bb] MacBook Pro graphics card options - what about Mavericks?

[Carlos Kikuti]

Hi,

Can I profit from Kristin's question and add one: is it too soon to know if the 
crystallography software (CCP4, Coot, Autobuster, XDS) will work fine with 
Mavericks (Mac OS X 10.9)? 

(I remember a bit of trouble when Lion came off).

Carlos

Em 23 oct. 2013, às 22:10, Kristin Low kristin@queensu.ca escreveu:

 Hi everyone,
 
 Sorry for being a bit off topic, but I thought this group would be great for 
 advice.
 
 I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn 
 as to whether I need integrated vs discrete graphics for structural biology, 
 including molecular modelling, especially since the latest advances by Intel 
 in terms of integrated graphics. Right now with the new releases, the options 
 are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia GT 750M. 
 Thoughts?
 
 
 Thanks everyone!
 
 Kristin
 
 
 -
 Kristin Low
 Ph.D. Candidate
 Queen's University
 Department of Biomedical and Molecular Sciences
 Botterell Hall, Room 645
 Kingston, ON
 Canada  K7L 3N6
 
 tel: +1-613-533-3019
 -
 

Re: [ccp4bb] Mac OS 10.8 Mountain Lion and CCP4?

[Carlos Kikuti]

Hi all,

On the same topic, I'd like to know wether the retina display is also
fine, because I've found complains about X11 (non crystallographic)
programs appearing ugly in it. Is anybody using it?

Just for info, I'm also using Mountain lion without problems ( Fink got
weird but I don't really care).

Carlos
De : Huw Jenkins
Envoyé : 21/02/2013 21:44
À : CCP4BB@jiscmail.ac.uk
Objet : Re: [ccp4bb] Mac OS 10.8 Mountain Lion and CCP4?
All works fine. You'll need to install XQuartz (Apple are no longer
supplying  their own X11 builds) but that's just a couple of clicks in
the box that pops up the first time you run an program that needs X.

The (on by default) versioning auto save is a PITA if you ever use
textedit to edit scripts but apart from that 10.8 is a fairly painless
upgrade.

Huw

Re: [ccp4bb] Problems in scaling up expression

[Carlos Kikuti]

I'm replying quite late, so you've probably solved the problem by now, but here 
it goes:

I agree with Shane, and I bet your procedure needs LESS aeration (15 ml in a - 
I guess - 50 ml Falcon doesn't leave much space for air, and Falcons are 
hermetically closed). In BL21 that's usually done for increasing solubility, 
despite of the lower expression level. I don't know your cells, maybe they 
behave differently for some reason.

Carlos


 
Em 15/01/2013, às 18:52, Shane Caldwell shane.caldwel...@gmail.com escreveu:

 Hi James,
 
 The first place I'd look is oxygen conc, as you'll get different aeration 
 from different vessel shapes/sizes. You could play around with shaking speed 
 and/or flask geometry to try to get more or less aeration. 
 
 Shane Caldwell
 McGill University
 
 On Tue, Jan 15, 2013 at 9:48 AM, Murray, James W j.w.mur...@imperial.ac.uk 
 wrote:
 Dear All.
 
 A question on protein expression.
 We have been doing small scale test expressions in 15ml of terrific
 broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching
 OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours
 at 37 C. There is a big band corresponding to our protein in the
 soluble fraction and not much in the insoluble fraction. However, on
 scaling up to 1 L TB cultures (with same concentrations of kanamycin
 and inducing with same concs of rhamnose+IPTG) we don't get strong
 over expression. Has anyone else experienced problems when scaling up
 expression? (and more importantly, solved them?)
 
 best wishes
 
 James
 
 
 --
 Dr. James W. Murray
 David Phillips Research  Fellow
 Division of Molecular Biosciences
 Imperial College, LONDON
 Tel: +44 (0)20 759 48895
 

Re: [ccp4bb] Fab Preparation

[Carlos Kikuti]

Hi Abdul

Yes, I had the same bad experience with some of the Fabs I was preparing by 
papain digestion several years ago. By the time I just walked around it by 
starting with more antibody, yields were less than 20% of the starting antibody 
mass (bad as compared to the 60% from the good ones).

I didn't try this myself, but I believe you can test different buffers for the 
digestion in small scale (varying salt concentration and pH - rather going up - 
a little bit). The loss in papain activity can be partially compensated by 
adding more enzyme. I wouldn't change the antibody nor the L-Cys concentrations.

Carlos


Em 24/12/2012, às 03:39, Abdul Khan escreveu:

 Hi everyone.
 We are trying to make Fabs from monoclonal IgGs, we are using insoluble 
 papain for digestion, to our surprise the protocol we were using gave 
 precipitates with some antibodies but not all, we have tried 4, Two were fine 
 and the other 2 just precipitated during the digestion. Our digestion 
 reaction contains following,
 50 mM Na-Acetate pH5.5, 1mM EDTA, 50mM L-cysteine, no salt, antibody 
 concentration 1-2 mg/ml.
 We appreciate if someone has such experience and would like to share some 
 expert views,
 thanks a lot,
 abdul
 
 -- 
 Abdul Ghafoor Khan 
 University of Vienna, 
 Inst. Med. Biochem., Vienna Biocenter (VBC), 
 Dr. Bohr Gasse 9/3, 
 A-1030 Vienna, Austria, 
 Tel: 0043 1 4277 61631,(lab) 
 Tel: 0043 69910695081, (mobile) 
 Email:abdul.ghaf...@univie.ac.at

Re: [ccp4bb] Salt bridge in crystallization

[Carlos Kikuti]

In think most of the salt bridges I recall in structures are either in the core 
of the protein or in interfaces (crystallin or complex interfaces with little 
or no polar solvent around). Just like charge interactions, the lower 
dielectric constant of the environment makes them stronger.

 Carlos


Em 20/10/2012, às 04:49, Ed Pozharski escreveu:

 On 10/19/2012 10:37 PM, Acoot Brett wrote:
 Will you please explain to me why the protein salt bridge can still exist in 
 the high salt concentration as used in the crystallization condition?
 
 You are saying it as if there is some fundamental law of nature that says 
 that salt bridges cannot be maintained at high salt concentration.  I know of 
 no such law.  The observation simply means that (mostly for entropic reasons) 
 the free energy of the salt bridge is still lower.  If you present your case 
 as to why the salt bridges must be disrupted, we can poke holes in it :)
 
 Cheers,
 
 Ed
 
 -- 
 Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
 Julian, King of Lemurs

Re: [ccp4bb] Tricky MR problem

[Carlos Kikuti]

That's good to know, Rhys.

But would you mind sharing why did it work with Balbes? Is there a big change 
in the position of the domains as related to your first searching model, or 
huge loops that had been removed?

Carlos



Em 02/10/2012, às 14:42, RHYS GRINTER escreveu:

 Thanks for your help everyone!
 
 It seems that the Balbes pipeline, followed by density modification in Phenix 
 has done the trick 
 
 Rhys

Re: [ccp4bb] Tricky MR problem

[Carlos Kikuti]

Not sure I understood your problem but it looks like it's related to 
flexibility. You can try cutting the domains apart (and chopping off the loops) 
in two different pdbs. They can be used as separate ensembles. Try to find the 
bigger one first. 35% homology is not that low, just search with polyAla.

(You can also analyze the analogue with Normal Modes to see where it bends in 
case it's not obvious).

Carlos


Em 01/10/2012, às 21:26, RHYS GRINTER escreveu:

 Hi All,
 
 I'm currently working on solving the structure of a protein by molecular 
 replacement. The protein is around 30kDa and likely has a two beta-prism 
 domains, linked by a long curved two stranded sheet based on the structure of 
 an analogue. There are also a number of other structures which represent a 
 single homologous beta-prism domain.
 I've tried to find MR solution using the analogue and various truncation/AA 
 substitution models based on it with no success. I've also tried single 
 domain ensembles of the other homologous structures, also with no success. I 
 think the problem is the overall sequence homology is quite low between my 
 protein and the available structures (35% for the analogue and around 20% for 
 the other models.
 
 I was curious as to how someone with more experience would tackle this 
 problem.
 
 Just for background, the datasets I have are 2 to 2.7 angstroms with pretty 
 nice stats. The space group is most likely C2221 with two molecules per ASU 
 (giving around 58% solvent).
 
 Thanks,
 
 Rhys Grinter
 PhD Candidate
 University of Glasgow

Re: [ccp4bb] off topic detection of oligomerisation

[Carlos Kikuti]

Urea?!?!? My arm hair went up.

Anyways, DLS (Dynamic Light Scattering) might help. Hard to imagine something 
quicker and easier, provided that someone next to you already has a DLS machine.


(sorry for the late answer)

Carlos



Em 26/06/2012, às 15:10, Brad Bennett escreveu:

 Native PAGE (i.e. BN-PAGE), light scattering (i.e. MALLS)
 
 Not quick and easy but could work: AUC (i.e. a sedimentation equilibrium 
 experiment)
 
 -Brad
 
 On Tue, Jun 26, 2012 at 9:01 AM, Careina Edgooms careinaedgo...@yahoo.com 
 wrote:
 Dear ccp4 bulletin board
 
 I apologise for off topic question. I wonder if anybody knows of a good 
 method to detect oligomerisation? 
 I suspect an equilibrium intermediate is forming oligomers based on 
 tryptophan fluorescence showing an exposed tryptophan becoming buried in a 
 hydrophobic region. I would like to perform some experiments to prove this. 
 Cross linking is not working for me and neither is SEC HPLC due to technical 
 issues with the urea I think.
 Are there any other quick and easy techniques that any of you can think of 
 off the cuff? Shifts in equilibrium unfolding curves done at different 
 concentrations will probably show something because of the law of mass action 
 but this is a big job needing many replicates so I wonder if there are any 
 other simple methods available?
 
 Thanks
 Careina
 

Re: [ccp4bb] Off topic: thermal melt

[Carlos Kikuti]

Hi Anita,

You don't state the WL of the melting and back folding CDs, I'm guessing it's 
around 210 nm? 

In any case, the refolding that you see might be just the formation of 
aggregates. For saying if it's really refolding you'd need a new spectrum WL vs 
AU in the end of the reverse thermal melt and superimpose it with the first 
one.

Having the protein more concentrated than that, or using a longer path length 
could reduce your noise.

Carlos



Em 14/05/2012, às 09:10, anita p escreveu:

 Hi All,
 Sorry for this off topic. 
 I am stuck with a plant protein which is not crystallizing. I am expressing 
 this in E coli.
 I have done the CD and thermal melt of the deletion constructs of this 
 protein @ 0.2 mg/ml.
 
 Please refer to the doc file attached to this email.
 
 The CD looks okay but the thermal melt looks kind of wierd. I would expect a 
 sigmoid curve for the thermal melts and then when we do a reverse temperature 
 scan, the protein should denature and may not fold back (with some exceptions)
 
 Please let me know if any one had the same experience before and could 
 explain me what this meant.
 
 Kind regards
 Anita
 Cd.doc

Re: [ccp4bb] recommendations_on_purification

[Carlos Kikuti]

90% of the protein could be aggregated and hiding the His tag from the resin. 
Metal ions might be removed from the starting sample by adding up to 10 mM EDTA 
in the first buffer exchange cycle, but just after reading 10% of the protein 
binds I wouldn't bet much on this. Rather increase the culture volume and move 
on.

Carlos Kikuti


Em 26/03/2012, às 14:39, Lorenzo Finci escreveu:

 Petros, 
 
 It has indeed been speculated that high concentrations of Magnesium and/or 
 other metals present in the cell lysate effect the binding of the 
 Histidine-tag, and thus specific conditions for binding and elution need to 
 be optimized for specific elution of your target protein. I believe that the 
 standard recommendations when using a Nickel column to bind to your 
 Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted 
 unspecific binding protein, to try manipulating the binding by lowering the 
 pH of the buffers, and by determining the specific concentration of . 
 Alternatively, you can also try using another metal column such as Cobalt 
 (Talon) with a higher affinity for the Histidine tag.
 Sincerely, 
 lorenzo
 
 
 Lorenzo Ihsan FInci, Ph.D.
 Postdoctoral Scientist, Wang Laboratory
 Harvard Medical School
 Dana-Farber Cancer Institute
 Boston, MA 
 Peking University
 The College of Life Sciences
 Beijing, China
 
 
 
 Date: Mon, 26 Mar 2012 15:04:43 +0300
 From: peg...@pasteur.gr
 Subject: [ccp4bb] recommendations_on_purification
 To: CCP4BB@JISCMAIL.AC.UK
 
 Dear all,
 
 I am expressing a 6xHis tagged secreted protein in a fermentor in P. 
 pastoris, using the standard minimal medium described in the invitrogen 
 manual (plus PTM1). Following collection of the culture medium, I am having 
 problems with purification of the protein as only a small fraction (~10%) 
 binds to the Ni-NTA beads even after extensive buffer exchange (when 
 expressed in full BMGY media this is not observed). Could this be attributed 
 to metal ions still present in my sample? Is it likely to be due to poor 
 protein quality in this medium? Or any other suggestions?
 
 Thanks in advance
 Petros 

Re: [ccp4bb] Na acetate as purification buffer

[Carlos Kikuti]

You might be giving too much importance to the THEORETICAL pI of the protein. 
If it's supposed to be well charged at pH 7,4 (only a titration curve, and not 
simply knowing the pI will tell you this)  and it's still precipitating, the 
problem might be due to a bad fold, for instance, or to the lack of salt ...

I've heard people saying that proteins with high pIs are more difficult to work 
with, but to be honest I don't know where that fear comes from.

Carlos

Em 02/03/2012, às 05:55, Artem Evdokimov escreveu:

 This pH is generally incompatible with Ni IMAC, sorry :) If you have a
 high pI your best bet is to employ ion exchange as primary capture,
 specifically SP resin or if you're really lucky - CM resin. There are
 only relatively few proteins in E. coli that bind to CM resin at pH 5
 and virtually none (one-three) that will bind at pH 8. If your protein
 still binds, then you're good to go.
 
 Artem
 
 On Thu, Mar 1, 2012 at 10:49 PM, anita p crystals...@gmail.com wrote:
 Hi all,
 Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein
 on Histrap column (AKTA) followed by SEC?
 My protein has a pI of 9. I tried pH7.4 but it has precipitation problems.
 While doing buffer screening using 24 well hanging drop I found that lower
 pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole
 purification???
 
 
  Thanks in advance
  Anita

Re: [ccp4bb] Fwd: [ccp4bb] protein degradation

[Carlos Kikuti]

I agree with Mark, except that I wouldn't even try sonication, Triton or 
freeze/thaw cycles in that case.

I'd look for emulsification (with a Homogenizer) in a cold room, but if you go 
quickly and gently with the French Press (either in a cold room or by using a 
cold piston) it might help. Don't use too much pressure, it heats up the sample.

I also agree that if it migrates as a single peak in gel filtration and in 
heparin sepharose, there is no reason for not setting some drops with it. And 
if you decide to do it, then simplify the purification and avoid submitting the 
protein to treatments that are not helping to get it purer. 

(I just found it weird that your fraction 6 has a huge load of protein , I 
guess those are actually the beads from the purification or something like 
that? In any case it seems to me that the fraction volume could be increased)

Carlos





Em 15/02/2012, às 16:39, Mark J van Raaij escreveu:

 try experimenting with different, especially protease-deficient, E coli 
 strains to express the protein and try different methods to lyse the bacteria 
 (sonication, french-press, emulsification, bead-beater, mortar  pestle under 
 liquid nitrogen).
 
 on the other hand, if you are lucky, you are just proteolysing some surface 
 loops and can still purify and crystallise the protein. This was done on 
 purpose for the cap-binding complex, see:
 Crystal structure of the human nuclear cap binding complex.
 Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S.
 Mol Cell. 2001 Aug;8(2):383-96.
 
 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij
 
 
 
 On 15 Feb 2012, at 14:09, Sivasankar Putta wrote:
 
 Dear All,
 
 Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi 
 domain) DNA binding protein, that we are expressing at 18 degree Centigrade 
 in E. Coli.  The protein appears to degrade during purification; we have 
 protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM 
 EDTA and 1mM DTT  throughout during purification ( right from lysis stage).  
  We handle the protein at 4 degree Centigrade.
 
 Can you please suggest what precautions we can try to avoid such degradation 
 ? 
 
 Please find the attached gel picture regarding protein
 
 Sivasankar Putta
 
 
 
 proteingel.pdf

Re: [ccp4bb] Mystery density

[Carlos Kikuti]

It's bang on the Y axis I'd just ignore it (treat it as just noise) and 
work on the rest of the model. There seems to be some trouble in other parts of 
it. (Had a similar case but at 3.5 A data).  I wonder what is the gap between 
Rfree and Rwork.

And I'm curious for what more experienced people will say about it.

Carlos


Em 01/12/2011, às 11:06, Mark J van Raaij escreveu:

 although it is hard to see in 3 2D views, some of the tentacles appear 
 connected to the protein, could they result from some of the long Lys or Arg 
 side-chains not following the 4-fold symmetry? i.e. alternative conformations?
 The density on the 4-fold may be ions, solvent, some metal ion? Noise 
 appears to collect on symmetry axes (for want of a better way to put it), so 
 the sigma-levels there are not very reliable. Sometimes one sees quite strong 
 negative peaks on symmetry axes that can not be explained, so I would expect 
 spurious positive peaks to also occur. Of course we normally just put a water 
 in it if it can make reasonable hydrogen bonds.
 
 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/content/research/macromolecular/mvraaij
 
 
 
 
 
 On 1 Dec 2011, at 10:36, Martin Montgomery wrote:
 
 Posting on behalf of Katia Baranova (see below).  Any suggestions gratefully 
 received!
 
 
 
 
 Hello,
 
 we have a puzzling density and hope someone has an idea of what it may be.
 
 The pictures of it are here:
 
 http://www.mrc-mbu.cam.ac.uk/sites/default/files/images/mystery/top.png
 http://www.mrc-mbu.cam.ac.uk/sites/default/files/images/mystery/front.png
 http://www.mrc-mbu.cam.ac.uk/sites/default/files/images/mystery/side.png
 
 The space group is I422, resolution 1.6A
 
 The trial contained Tris, DTT, EDTA, sodium, potassium, phosphate, and 
 chloride.
 
 The centre of our unknown feature is about 3.8A from the Arg and it is also 
 on the four fold symmetry axis.
 It is + 28 sigma in the center and  about + 9.5 sigma in each of the 
 tentacles.
 
 Thank you very much!
 
 Katia.
 
 
 
 On 30 Nov 2011, at 17:30, Katia Baranova wrote:
 
 Hello,
 
 Well, it doesn't work.
 Can you forward it please?
 
 thanks a lot,
 
 K
 
  Original Message 
 Subject:Rejected posting to CCP4BB@JISCMAIL.AC.UK
 Date:   Wed, 30 Nov 2011 17:27:45 +
 From:   JISCMAIL LISTSERV Server (16.0) lists...@jiscmail.ac.uk
 To: Katia Baranova e...@mrc-mbu.cam.ac.uk
 
 
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 the
 
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Re: [ccp4bb] gst-tag protein purify problem

[Carlos Kikuti]

What happens if you load your elution fraction into a size exclusion column? If 
your protein of interest comes in the void volume together with most of its 
contaminants, you'd better test a different construct, and that's much more 
than only changing the tag from N- to C-terminus. Sumo and GST might be 
solubilizing things that shouldn't really be soluble...

Carlos



Em 06/08/2011, às 16:43, Paul Kraft escreveu:

 Hi Lisa,
 Have you compared your yield of purified protein of soluble protein per gram 
 of pellet,  4M Guanidine solubilized pellet (wash and elute from Ni column 
 with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total 
 protein in the pellet on a page gel?   The main reason I would think you get 
 high background of cellular proteins on you Ni purification is because your 
 yield is too low (I know obvious..). There are a variety of methods to 
 increase yield like expressing at RT (assuming your expressing in E.coli), or 
 switching to yeast, insect, or mamallian cells if your protein is of human 
 origin. The most helpful and easiest  method to try first (after trying low 
 temp), would be switching the tag from N to C terminus or vice versa. 
 Otherwise switch to a thermophile version of your protein if possible (and 
 try cysteine mutants or other bacterial organisms for source the source gene).
 Paul
 ps one thing I forgot, you mention it is a DNA binding protein, solublizing 
 in 2M NaCl is much better than 1M NaCl, you could just be losing it...the 
 protein that is :-)
  
 Dr. Paul Kraft
 Structural Biologist
 cell 586-596-2770
 email: haresea...@yahoo.com
 email: kraft_proteome_resea...@yahoo.com
 
 
 
 
 This communication and any attachments contain information which is 
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 is taken to accept the risks in doing so.
 From: LISA science...@gmail.com
 To: CCP4BB@JISCMAIL.AC.UK
 Sent: Thursday, August 4, 2011 11:51 AM
 Subject: [ccp4bb] gst-tag protein purify problem
 
 hi guys,
 I have a DNA binding protein and expressed the DNA binding domain (150 aa) 
 with his-sumo tag or gst tag at the n-terminal. I tried to purified it with 
 Ni column or Gst column separately. But purity is lower than 50% after Ni or 
 GST column. This protein only stable with 1M Nacl or higher. I worked on it 
 almost half year. But I still can not get the pure protein.
 please give me some suggestions. Thank you.
  
 Lisa
 
 

Re: [ccp4bb] Displaying repeats in an amino acid sequence

[Carlos Kikuti]

Hi, there is a dot plot function in Geneious (www.geneious.com) that  
may be useful when the repeats are short.


Carlos Kikuti


Le 5 juin 10 à 01:00, CCP4BB automatic digest system a écrit :



On Fri, Jun 04, 2010 at 08:15:03PM +, Klaus Sengstack wrote:

Dear all,

I am looking for an alignment program that is able to display  
repeating units in the sequence. In the C-terminus of my protein I  
found some specific repeats which I want to visualize.  Does  
anybody know such program? Thanks a lot.


Best regards,
K.Sengstack




--

Re: [ccp4bb] protein monitoring

[Carlos Kikuti]

 4. protein monitoring


Dear Yogesha,

You can use the absorbance at 215 or 220 nm to follow your peptide  
during purification. Compounds like DTT and EDTA can increase the  
noise at those wavelengths, go to 220 nm if you have things like that  
in your purification buffers.


Quantification of the peptide will have to be performed by Bradford or  
other colorimetric method, and I guess you can use that quantification  
to find out the extinction coefficient of your peptide at 215-220 nm.


Luck,

Carlos