Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Srivastava, Dhiraj]

Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



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Re: [ccp4bb] [External] Re: [ccp4bb] Crystallizing a tough target

[Srivastava, Dhiraj]

While I understand that you want to have protein concentration at its 
solubility limit, i had several proteins which can go to 60-80 mg/ml, they 
seems to get crystallized at 10-15 mg/ml. All you want is saturation  in 
crystallization conditions.
Dhiraj srivastava


From: CCP4 bulletin board  on behalf of Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, February 5, 2024 5:32 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Crystallizing a tough target

Limited proteolytic might help, too.

https://www.nature.com/articles/nmeth1118


Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile



 Original Message 
On 5 Feb 2024, 10:27, kavyashreem < kavyashr...@instem.res.in> wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




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Re: [ccp4bb] [External] Re: [ccp4bb] Relion issue with MPI

[Srivastava, Dhiraj]

It was reported by me but the problem was not solved. I thought ccp4bb has much 
bigger user base and may be someone has experienced this issue.
It is something related to os (rocky linux) or mpi in my computer. Data set is 
not a problem as i can process same data set with exactly same parameters on a 
much less powerful computer without any problem. Also 2D classification on my 
computer is using gpu without any problem. Its only mpi processes of relion 
which are failing. Cryosparc is not a problem either.
Thanks
Dhiraj

From: Takanori Nakane 
Sent: Friday, December 22, 2023 5:35 PM
To: Srivastava, Dhiraj 
Cc: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Relion issue with MPI

Hi,

First of all, please report details of your hardware and your job.

- Type of GPU
- Number of GPU
- GPU memory size
- Box size
- Number of threads
- Number of MPI processes
- Full command line

Do you get the same error in ALL datasets (including our
tutorial dataset) or only on this particular dataset?

A very similar issue was reported in
https://github.com/3dem/relion/issues/1056
but I do not know what is the cause at the moment.

Best regards,

Takanori Nakane

On 12/23/23 03:31, Srivastava, Dhiraj wrote:
> Hi
> I am trying to use relion and I am getting error when trying to use mpi
> (for 3d classification and 3D auto-refine).
>
>
> ERROR: out of memory in
> /home/lvantol/relion5/relion/src/acc/cuda/custom_allocator.cuh at line
> 436 (error-code 2)
>
> in: /home/lvantol/relion5/relion/src/acc/cuda/cuda_settings.h, line 65
>
> ERROR:
>
> A GPU-function failed to execute.
>
>
> 2D classification is working fine with significant GPU usage. I tried 3
> different versions (4, 4 beta and 5 beta), one installed by vendor
> (Exxact) and all have the same issue.  I am able to do 3D auto-refine
> and 3D classification on the same data set using our cluster without any
> problem.  did anyone encounter a similar issue earlier? How can I fix
> this problem?
>
>
> Thank you
>
> Dhiraj
>
>
>
> 
>
> 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] Relion issue with MPI

[Srivastava, Dhiraj]

Hi
I am trying to use relion and I am getting error when trying to use mpi (for 3d 
classification and 3D auto-refine).


ERROR: out of memory in 
/home/lvantol/relion5/relion/src/acc/cuda/custom_allocator.cuh at line 436 
(error-code 2)

in: /home/lvantol/relion5/relion/src/acc/cuda/cuda_settings.h, line 65

ERROR:

A GPU-function failed to execute.


2D classification is working fine with significant GPU usage. I tried 3 
different versions (4, 4 beta and 5 beta), one installed by vendor (Exxact) and 
all have the same issue.  I am able to do 3D auto-refine and 3D classification 
on the same data set using our cluster without any problem.  did anyone 
encounter a similar issue earlier? How can I fix this problem?


Thank you

Dhiraj




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[ccp4bb] RMSF calculation in pymol

[Srivastava, Dhiraj]

Hi All
sorry for off topic question. does anyone have script for rmsf 
calculation of multistate pdb file in pymol? There used to be a script 
rmsf_states  written by Robert Campbell but with newer version of pymol, it's 
not working.

Thank you
Dhiraj



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Re: [ccp4bb] [External] Re: [ccp4bb] Structure prediction - waiting to happen

[Srivastava, Dhiraj]

That’s the point. All these predictions can not replace experiments and they 
should be used only in the absence of experimental structures, that too with 
caution. A biophysicist and structural biologist understand this but most of 
the non-experts (including the reviewers of the grants from non-structural 
biology study sections) don’t understand this and think that with alphafold, 
there is no need for experimentally determined structures. That’s more damaging 
than helpful.



From: CCP4 bulletin board  on behalf of Eugene Valkov 

Sent: Sunday, April 2, 2023 10:10 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] Re: [ccp4bb] Structure prediction - waiting to 
happen


Predicted structures lack the precision and accuracy of 
experimentally-determined structures at high resolution with all the benefits 
of unbiased co-discovery of solvent molecules, ions, ligands, etc., bound to 
molecules of interest.


It is also true that AI-assisted structure prediction can be stunningly 
accurate to the level of correctly identifying interfacing residues between 
interacting proteins and accurately recapitulating the architectures of large, 
multi-domain complexes. AI-assisted structure prediction is immensely 
liberating in lowering the threshold to generate testable hypotheses for those 
lacking access to experimental structural biology resources.


AlphaFold and related structure prediction tools are here to stay, and no magic 
incantations in funding proposals will likely make them disappear. So rather 
than wring our hands as a community and mourning the loss of 
‘divide-and-conquer’ structural biology, which was a rich vein to mine over the 
last decades, we should adapt to structure prediction and normalize its use, 
with proper controls and caveats, as part of our arsenal of tools and methods 
to focus on the questions under investigation.


With best wishes,

Eugene


Eugene Valkov, D.Phil.

Stadtman Investigator

RNA Biology Laboratory

Center for Cancer Research

National Cancer Institute

Frederick MD 21702, USA

(301) 846-1823


On Sun, 2 Apr 2023 at 10:44, jacinto.ls<http://jacinto.ls> 
mailto:jlopez.sagas...@gmail.com>> wrote:

I am also not sure whether AlphaFold can address the impact of ions and other 
cofactors on the fold of many proteins.

Best wishes,
Jacinto

On 2/4/23 16:20, Srivastava, Dhiraj wrote:
May be this article is of some help suggesting the need of experimental 
structures despite excellent alphafold model.
https://www.nature.com/articles/s41401-022-00938-y


From: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
on behalf of Ian Tickle <mailto:ianj...@gmail.com>
Sent: Sunday, April 2, 2023 8:28 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [External] Re: [ccp4bb] Structure prediction - waiting to happen


All, the first hurdle will of course be whether the AlphaFold model works as a 
MR model, even with the 100% completeness and sequence identity of a bespoke 
model.  The question is what B factors to use or which disordered bits to leave 
out, as that can strongly influence the result (perhaps use info from a similar 
structure).  If it doesn't work in MR that's a pretty good indication that it's 
too far from reality to be useful for looking at detailed interactions.

Does anyone know of a systematic investigation of the success rate of AlphaFold 
models in MR ?  That would be useful ammunition !

Cheers

-- Ian


On Sun, 2 Apr 2023 at 11:51, Gerard Bricogne 
mailto:g...@globalphasing.com>> wrote:
Dear all,

 I think that quoting general viewpoints and statements, however
knowledgeable and respected their authors may be, will only exacerbate the
climate of clashing prejudices between two camps and is bound to sustain a
war of opinions rather than lead to a rational acceptance that something has
changed. The frustration is that one camp (the AlphaFold believers) can be
viewed as in effect preventing experiments that could prove it wrong.

 One way to deal with this obstruction would be to provide, in each
particular case, evidence that the AlphaFold results "do not cut it" as the
sole provider of 3D information within the project at hand. This means that
every grant proposal requesting resources towards a crystallographic
structure solution should document the fact that AlphaFold predictions have
been performed (or, often, looked up in a database of pre-cooked results)
but do not provide the accuracy required for the proposed investigation. If
this step of writing up the "Background" section of the grant actually
delivers a useful result, then everyone will be happy; and if it doesn't,
then the case for the need to allocate resources to solving the structure by
crystallography will be unassailable. In this way, AlphaFold will be a game
changer (we have known that since July 202

Re: [ccp4bb] [External] Re: [ccp4bb] Structure prediction - waiting to happen

[Srivastava, Dhiraj]

May be this article is of some help suggesting the need of experimental 
structures despite excellent alphafold model.
https://www.nature.com/articles/s41401-022-00938-y


From: CCP4 bulletin board  on behalf of Ian Tickle 

Sent: Sunday, April 2, 2023 8:28 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Structure prediction - waiting to happen


All, the first hurdle will of course be whether the AlphaFold model works as a 
MR model, even with the 100% completeness and sequence identity of a bespoke 
model.  The question is what B factors to use or which disordered bits to leave 
out, as that can strongly influence the result (perhaps use info from a similar 
structure).  If it doesn't work in MR that's a pretty good indication that it's 
too far from reality to be useful for looking at detailed interactions.

Does anyone know of a systematic investigation of the success rate of AlphaFold 
models in MR ?  That would be useful ammunition !

Cheers

-- Ian


On Sun, 2 Apr 2023 at 11:51, Gerard Bricogne 
mailto:g...@globalphasing.com>> wrote:
Dear all,

 I think that quoting general viewpoints and statements, however
knowledgeable and respected their authors may be, will only exacerbate the
climate of clashing prejudices between two camps and is bound to sustain a
war of opinions rather than lead to a rational acceptance that something has
changed. The frustration is that one camp (the AlphaFold believers) can be
viewed as in effect preventing experiments that could prove it wrong.

 One way to deal with this obstruction would be to provide, in each
particular case, evidence that the AlphaFold results "do not cut it" as the
sole provider of 3D information within the project at hand. This means that
every grant proposal requesting resources towards a crystallographic
structure solution should document the fact that AlphaFold predictions have
been performed (or, often, looked up in a database of pre-cooked results)
but do not provide the accuracy required for the proposed investigation. If
this step of writing up the "Background" section of the grant actually
delivers a useful result, then everyone will be happy; and if it doesn't,
then the case for the need to allocate resources to solving the structure by
crystallography will be unassailable. In this way, AlphaFold will be a game
changer (we have known that since July 2021) but not a game killer. Savvas
alluded to a similar approach, but it could be made a formal requirement
acceptable to both proposers and reviewers, who would then both be dealing
with the situation in a scientific rather than dogmatic manner.


 With best wishes,

  Gerard.

--
On Sat, Apr 01, 2023 at 06:54:33PM +, Goldman, Adrian wrote:
> I think this is all true - and I’ve been putting things like this into my 
> (failing) grants - but I get the dispiriting sense that the medics think (to 
> borrow a line from hamlet) “the applicant doth protest too much methinks”.
>
> Well if as per James H today ;), we deposit coordinates to 1sf, alphafold 
> will be just fine.
>
> Of course the coordinates won’t be of any use to anybody, but the pictures 
> will be nice.
>
> Adrian
>
> Sent from my iPhone
>
> > On 1 Apr 2023, at 21:39, Randy John Read 
> > mailto:rj...@cam.ac.uk>> wrote:
> >
> > There’s also this preprint with Tom Terwilliger as lead author: 
> > https://www.biorxiv.org/content/10.1101/2022.11.21.517405v1. The title is 
> > “AlphaFold predictions: great hypotheses but no match for experiment”.
> >
> > Best wishes,
> >
> > Randy
> >
> >> On 1 Apr 2023, at 18:18, Savvas Savvides 
> >> <9d24f7f13e09-dmarc-requ...@jiscmail.ac.uk>
> >>  wrote:
> >>
> >> Dear Rams,
> >>
> >> I salute you for sharing this.
> >>
> >> Just a week ago, I also received a remark along these lines on a declined 
> >> grant application. The remark was the only unfavourable point, which 
> >> suggested that it must have weighed disproportionally towards the negative 
> >> outcome. This was a two-stage evaluation process and the grant was cut in 
> >> stage-1 where it was evaluated by a small group of evaluators, none of 
> >> whom was a structural biologist/biochemist. Stage-2 would have involved 
> >> peer review by international experts.
> >>
> >> Despite my initial disbelief about what this remark might have caused and 
> >> upon reflection, I realized that it might be time to become proactive in 
> >> future applications in anticipation of the apparent growing trend towards 
> >> such remarks and perceptions.
> >>
> >> I think that a generalized form of preemptive text might not serve the 
> >> purpose well, but perhaps well-articulated statements specific to the 
> >> proposed biological problem at hand (perhaps aided by illustrations 
> >> demonstrating the inability of structure prediction to address the problem 
> >> at hand) might be the better way to go. Even though many of us 

Re: [ccp4bb] [External] Re: [ccp4bb] Structure prediction - waiting to happen

[Srivastava, Dhiraj]

No offense to anyone but most of these systematic studies are often on small 
not so flexible, single domain, easy to crystallize proteins with little 
conformational variability and that’s where alpha fold excel. It fails with 
(may not be all though) multidomain proteins with conformational variability, 
and most of the systematic studies often exclude these tricky cases.



From: CCP4 bulletin board  on behalf of Ian Tickle 

Sent: Sunday, April 2, 2023 8:28 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Structure prediction - waiting to happen


All, the first hurdle will of course be whether the AlphaFold model works as a 
MR model, even with the 100% completeness and sequence identity of a bespoke 
model.  The question is what B factors to use or which disordered bits to leave 
out, as that can strongly influence the result (perhaps use info from a similar 
structure).  If it doesn't work in MR that's a pretty good indication that it's 
too far from reality to be useful for looking at detailed interactions.

Does anyone know of a systematic investigation of the success rate of AlphaFold 
models in MR ?  That would be useful ammunition !

Cheers

-- Ian


On Sun, 2 Apr 2023 at 11:51, Gerard Bricogne 
mailto:g...@globalphasing.com>> wrote:
Dear all,

 I think that quoting general viewpoints and statements, however
knowledgeable and respected their authors may be, will only exacerbate the
climate of clashing prejudices between two camps and is bound to sustain a
war of opinions rather than lead to a rational acceptance that something has
changed. The frustration is that one camp (the AlphaFold believers) can be
viewed as in effect preventing experiments that could prove it wrong.

 One way to deal with this obstruction would be to provide, in each
particular case, evidence that the AlphaFold results "do not cut it" as the
sole provider of 3D information within the project at hand. This means that
every grant proposal requesting resources towards a crystallographic
structure solution should document the fact that AlphaFold predictions have
been performed (or, often, looked up in a database of pre-cooked results)
but do not provide the accuracy required for the proposed investigation. If
this step of writing up the "Background" section of the grant actually
delivers a useful result, then everyone will be happy; and if it doesn't,
then the case for the need to allocate resources to solving the structure by
crystallography will be unassailable. In this way, AlphaFold will be a game
changer (we have known that since July 2021) but not a game killer. Savvas
alluded to a similar approach, but it could be made a formal requirement
acceptable to both proposers and reviewers, who would then both be dealing
with the situation in a scientific rather than dogmatic manner.


 With best wishes,

  Gerard.

--
On Sat, Apr 01, 2023 at 06:54:33PM +, Goldman, Adrian wrote:
> I think this is all true - and I’ve been putting things like this into my 
> (failing) grants - but I get the dispiriting sense that the medics think (to 
> borrow a line from hamlet) “the applicant doth protest too much methinks”.
>
> Well if as per James H today ;), we deposit coordinates to 1sf, alphafold 
> will be just fine.
>
> Of course the coordinates won’t be of any use to anybody, but the pictures 
> will be nice.
>
> Adrian
>
> Sent from my iPhone
>
> > On 1 Apr 2023, at 21:39, Randy John Read 
> > mailto:rj...@cam.ac.uk>> wrote:
> >
> > There’s also this preprint with Tom Terwilliger as lead author: 
> > https://www.biorxiv.org/content/10.1101/2022.11.21.517405v1. The title is 
> > “AlphaFold predictions: great hypotheses but no match for experiment”.
> >
> > Best wishes,
> >
> > Randy
> >
> >> On 1 Apr 2023, at 18:18, Savvas Savvides 
> >> <9d24f7f13e09-dmarc-requ...@jiscmail.ac.uk>
> >>  wrote:
> >>
> >> Dear Rams,
> >>
> >> I salute you for sharing this.
> >>
> >> Just a week ago, I also received a remark along these lines on a declined 
> >> grant application. The remark was the only unfavourable point, which 
> >> suggested that it must have weighed disproportionally towards the negative 
> >> outcome. This was a two-stage evaluation process and the grant was cut in 
> >> stage-1 where it was evaluated by a small group of evaluators, none of 
> >> whom was a structural biologist/biochemist. Stage-2 would have involved 
> >> peer review by international experts.
> >>
> >> Despite my initial disbelief about what this remark might have caused and 
> >> upon reflection, I realized that it might be time to become proactive in 
> >> future applications in anticipation of the apparent growing trend towards 
> >> such remarks and perceptions.
> >>
> >> I think that a generalized form of preemptive text might not serve the 
> >> purpose well, but perhaps well-articulated statements specific to the 
> >> proposed 

Re: [ccp4bb] [External] Re: [ccp4bb] Structure prediction - waiting to happen

[Srivastava, Dhiraj]

When I tested alpha fold on some of my proteins, it failed to predict the 
intramolecular interactions needed for their functions. Alphafold predicts the 
folds and overall structure of single domain and may be simple multi domain 
proteins but when conformational changes are needed for protein function, it 
fails to predict that. At-least it was the situation with some of my proteins. 
May be if somehow biochemical constraints are applied, it may predict the 
structure but you can not rely on alphafold to understand molecular mechanism 
of protein function. It’s too premature to think that alphafold can replace the 
need for experimental data.

Dhiraj



From: CCP4 bulletin board  on behalf of Ian Tickle 

Sent: Saturday, April 1, 2023 9:46 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Structure prediction - waiting to happen


Hi Ramaswamy

I assume this is an April Fool's but it's still a serious question because many 
reviewers who are not crystallographers or electron microscopists may not fully 
appreciate the difference currently between the precision of structures 
obtained by experimental and predictive methods, though the latter are 
certainly catching up.  The answer of course lies in the mean co-ordinate 
precision, related to the map resolution.

Quoting https://people.cryst.bbk.ac.uk/~ubcg05m/precgrant.html :

"The accuracy and precision required of an experimentally determined model of a 
macromolecule depends on the biological questions being asked of the structure. 
 Questions involving the overall fold of a protein, or its topological 
similarity to other proteins, can be answered by structures of fairly low 
precision such as those obtained from very low resolution X-ray crystal 
diffraction data [or AlphaFold].  Questions involving reaction mechanisms 
require much greater accuracy and precision as obtained from well-refined, 
high-resolution X-ray structures, including proper statistical analyses of the 
standard uncertainties (s.u.'s) of atomic positions and bond lengths.".

According to https://www.nature.com/articles/s41586-021-03819-2 :

The accuracy of AlphaFold structures at the time of writing (2021) was around 
1.0 Ang. RMSD for main-chain and 1.5 Ang. RMSD for side-chain atoms and 
probably hasn't changed much since.  This is described as "highly accurate"; 
however this only means that AlphaFold's accuracy is much higher in comparison 
with other prediction methods, not in comparison with experimental methods.  
Also note that AlphaFold's accuracy is estimated by comparison with the X-ray 
structure which remains the "gold standard"; there's no way (AFAIK) of 
independently assessing AlphaFold's accuracy or precision.

Quoting https://scripts.iucr.org/cgi-bin/paper?S0907444998012645 :

"Data of 0.94 A resolution for the 237-residue protein concanavalin A are used 
in unrestrained and restrained full-matrix inversions to provide standard 
uncertainties sigma(r) for positions and sigma(l) for bond lengths. sigma(r) is 
as small as 0.01 A for atoms with low Debye B values but increases strongly 
with B."

There's a yawning gap between 1.0 - 1.5 Ang. and 0.01 Ang.!  Perhaps AlphaFold 
structures should be deposited using James Holton's new PDB format (now that is 
an April Fool's !).

One final suggestion for a reference in your grant application: 
https://www.biorxiv.org/content/10.1101/2022.03.08.483439v2 .

Cheers

-- Ian


On Sat, 1 Apr 2023 at 13:06, Subramanian, Ramaswamy 
mailto:subra...@purdue.edu>> wrote:
Dear All,

I am unsure if all other groups will get it - but I am sure this group will 
understand the frustration.

My NIH grant did not get funded.  A few genuine comments - they make excellent 
sense.  We will fix that.

One major comment is, “Structures can be predicted by alpfafold and other 
software accurately, so the effort put on the grant to get structures by X-ray 
crystallography/cryo-EM is not justified.”

The problem is when a company with billions of $$s develops a method and blasts 
it everywhere - the message is so pervasive…

Question: Is there a canned consensus paragraph that one can add with 
references to grants with structural biology (especially if the review group is 
not a structural biology group) to say why the most modern structure prediction 
programs are not a substitute for structural work?

Thanks.


Rams
subra...@purdue.edu






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This message was 

Re: [ccp4bb] [External] Re: [ccp4bb] Missing double bonds in the ligand

[Srivastava, Dhiraj]

You can make molecule in chemdraw or chemdraw 3d with proper bond order and 
save it in sdf or .mol format or any other format compatible with schrodinger. 
Schrodinger should be able to read .sdf or .mol format.

Dhiraj



From: CCP4 bulletin board  on behalf of Nigel Moriarty 

Sent: Thursday, July 7, 2022 7:30 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Missing double bonds in the ligand

Joel

You'll need to be more specific than ".cif" files. CIF can be used for models, 
data and restraints. Read more here.

http://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2013_01.pdf#page=6

If you are referring to the restraints CIF, it is possible but I'm not sure 
which software uses it.

Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Phone : 510-486-5709 Email : nwmoria...@lbl.gov
Fax   : 510-486-5909  Web  : CCI.LBL.gov
ORCID : orcid.org/-0001-8857-9464


On Thu, Jul 7, 2022 at 3:06 PM Joel Tyndall 
mailto:joel.tynd...@otago.ac.nz>> wrote:

Does the Schrodinger software read a .cif file? The bond order should be 
embedded in there.



From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Nigel Moriarty
Sent: Friday, 8 July 2022 7:47 am
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Missing double bonds in the ligand



Further reading



https://en.wikipedia.org/wiki/The_Treachery_of_Images



https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2016_01.pdf#page=10


Cheers



Nigel



---

Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging

Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Phone : 510-486-5709 Email : nwmoria...@lbl.gov
Fax   : 510-486-5909  Web  : 
CCI.LBL.gov

ORCID : 
orcid.org/-0001-8857-9464





On Thu, Jul 7, 2022 at 12:42 PM Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:

Dear Anil,



Bond orders are not captured in PDB files explicitly. The way bonds are shown 
depends on the program and possibly additional information sources (i.e. 
molecular restraint or topology files).

So this is just a "problem" with Schrödinger.



Cheers,

Robbie



On 7 Jul 2022 21:00, "Dr. Anil Kumar Marapaka" 
mailto:anilmarap...@gmail.com>> wrote:

Dear CCP4 Users,

We are working on a ligand-bound structure and would like to look at 
protein-ligand interactions using Schrodinger.

Molecule structure is:

When we load the ligand-fitted PDB file into Schrodinger, the molecule appears 
to be flat like it should be in the structure but it removed all the double 
bonds in the ligand where the above shown original molecular structure has 
double bonds in it.

When we load the same PDB file into pymol and look at the ligand it shows 
correctly



We would appreciate if any insight or suggestions for why this may be happening 
and how to ensure the ligand shows properly when the PDB is loaded into 
Schrodinger. Would this be an issue with Schrodinger software or with the PDB 
file?

I can be available by direct email for troubleshooting at 

[ccp4bb] Baculovirus expression system

[Srivastava, Dhiraj]

Thank you every one for all your input. Our concern was whether there is any 
new advanced system than Bac-to-Bac or Bac-to-Bac is still pretty standard 
system for baculovirus expression. Based on all your inputs, it appears that we 
are not very outdated in our information about baculovirus expression system. 
There may be few newer systems but not much advanced than Bac-to-Bac and don't 
save significant amount of time and effort.

Thank you
Dhiraj



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[ccp4bb] Baculovirus expression system

[Srivastava, Dhiraj]

Hi All
 sorry for the question not related to crystallography. What is the 
baculovirus expression system that people use these days to get good yield in 
less time?

Thank you
Dhiraj



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Re: [ccp4bb] [External] Re: [ccp4bb] protease inhibitor and 3c protease

[Srivastava, Dhiraj]

Thanks Nikolay
 I am doing on column cleavage, so I am incubating it 
overnight at 4-degree C. Do you think even on column digestion is also going to 
be done in one hour.

Thank you
Dhiraj

From: CCP4 bulletin board  on behalf of Nikolay Dobrev 

Sent: Thursday, February 3, 2022 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease

Dear Dhiraj,
the 3C protease is from the Cysteine protease family.
Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC 
purification) and that will inhibit most of the metalloprotease. Furthermore, 
you can resort to Serine type protease inhibitors like PMSF or Aprotinin.

One side not, the 3C cleavage does not need to be long, as it is an extremely 
potent protease. If the cleavage sequence is well accessible in less than 1 
hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to 
substrate. Increasing the temperature speeds up the cleavage dramatically and 
at room temperature, you can obtain the same efficiency for 10 mins approx.
I am assuming you are interested in using the 3C protease to cleave a tag from 
recombinantly expressed protein.


Kind regards,
Nikolay

On 02/03/2022 6:25 PM Srivastava, Dhiraj  wrote:


Hi
sorry for off topic question. does anyone know which protease inhibitors we 
can include safely while cleaving with 3C protease?

Thank you
Dhiraj



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Nikolay Dobrev
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents<https://twitter.com/emblevents> | 
facebook.com/embl.org<http://facebook.com/embl.org> | 
youtube.com/user/emblmedia<http://youtube.com/user/emblmedia>
Visit www.embl.org/events<http://www.embl.org/events> for a complete list of 
all EMBL events.




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[ccp4bb] protease inhibitor and 3c protease

[Srivastava, Dhiraj]

Hi
sorry for off topic question. does anyone know which protease inhibitors we 
can include safely while cleaving with 3C protease?

Thank you
Dhiraj



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Re: [ccp4bb] [External] Re: [ccp4bb] protein DNA complex structure and extension of DNA structure

[Srivastava, Dhiraj]

Thank for the suggestion.
I think I should have been more specific about the problem. The promoter has 
binding site for two different transcription factors which interacts with each 
other to affect the transcription in spatiotemporal manner. I solved the 
structure of one protein (not deposited in PDB yet) which shows DNA bending. I 
am curious to see how this DNA bending affect the conformation of DNA nearby 
thus potentially affecting the binding of other transcription factor through 
DNA mediated allosterism. Off-course I am going to do simulation after 
extending the DNA. Since DNA is bend, I can not simply extend it using ideal 
DNA double helix conformation. How can I extend it computationally? x3DNA will 
work but needs pdb id.


Thanks
Dhiraj

From: Phoebe A. Rice 
Sent: Saturday, January 1, 2022 11:01 AM
To: Srivastava, Dhiraj ; CCP4BB@JISCMAIL.AC.UK 

Subject: [External] Re: [ccp4bb] protein DNA complex structure and extension of 
DNA structure


I would try the tools on this site - http://web.x3dna.org/index.php/protein





From: CCP4 bulletin board  on behalf of "Srivastava, 
Dhiraj" 
Reply-To: "Srivastava, Dhiraj" 
Date: Monday, December 27, 2021 at 8:32 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] protein DNA complex structure and extension of DNA structure



Hi

I solved the structure of protein DNA complex where the DNA molecule is 
bent. I want to extend the DNA computationally so I can model two DNA binding 
proteins together on same DNA molecule. is there a way by which I can extend 
the DNA in both direction? its easy to do it with ideal DNA molecule but I 
don't know how to do it with bent DNA.



Thank you

Dhiraj





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[ccp4bb] protein DNA complex structure and extension of DNA structure

[Srivastava, Dhiraj]

Hi
I solved the structure of protein DNA complex where the DNA molecule is 
bent. I want to extend the DNA computationally so I can model two DNA binding 
proteins together on same DNA molecule. is there a way by which I can extend 
the DNA in both direction? its easy to do it with ideal DNA molecule but I 
don't know how to do it with bent DNA.

Thank you
Dhiraj



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Re: [ccp4bb] [External] [ccp4bb] High-order oligomers vs robust crystals - references?

[Srivastava, Dhiraj]

Recently there were few articles where synthetic symmetrization was used to 
enhance the crystallizability. proteins used were MBP, lysozyme and GFP to name 
a few. you can search for it. one of them is -
https://www.sciencedirect.com/science/article/pii/S0969212615002890

[https://ars.els-cdn.com/content/image/1-s2.0-S0969212615002890-fx1.jpg]
A Suite of Engineered GFP Molecules for Oligomeric 
Scaffolding
Applications ranging from synthetic biology to protein crystallization could be 
advanced by facile systems for connecting multiple proteins together i…
www.sciencedirect.com


Dhiraj

From: CCP4 bulletin board  on behalf of Frank von Delft 

Sent: Friday, November 12, 2021 8:52 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] High-order oligomers vs robust crystals - 
references?

Hello all

Two decades ago, I remember (!) much talk about a reason that bacterial
proteins crystallize "more easily" is that they tend to come as
oligomers (dimers and up), and that this internal symmetry made them
happier to crystallize.

Did anybody ever publish hard evidence?  Or even, is there a primary
citation for the idea?

Thanks
Frank



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[ccp4bb] postdoctoral position available

[Srivastava, Dhiraj]

I am posting this advertisement on behalf of Prof. Nikolai Artemyev.


A postdoctoral position is available immediately at the Department of Molecular 
Physiology and Biophysics, University of Iowa Carver College of Medicine to 
study the molecular mechanisms of vertebrate visual transduction. We are using 
multiple approaches, including protein structure-function analysis, 
biochemistry, structural and molecular biology. A successful applicant will 
have a PhD, experience in biochemistry/structural and molecular biology, and a 
relevant publication record. Experience in x-ray crystallography and/or cryo-EM 
is preferred. Salary is commensurate with experience.

The University of Iowa is located in Iowa City, a dynamic college-town within 
driving distance of Chicago, St. Louis, and Minneapolis. Iowa City offers a 
culturally rich and highly academic environment supportive of the arts and 
sciences. The University of Iowa is part of the Molecular Biology Consortium 
that gives us substantial beam time for crystallographic studies at the 4.2.2 
ALS beamline (Lawrence Berkeley National Labs). We have active proposals at the 
Pacific Northwest Cryo-EM Center (PNCC) in Portland, OR.


Interested applicants should send a summary of previous research and a CV with 
names of at least two referees by e-mail to 
nikolai-artem...@uiowa.edu



Thank you
Dhiraj



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Re: [ccp4bb] [External] [ccp4bb] Add hydrogens

[Srivastava, Dhiraj]

Even pymol and chimera can add hydrogen. Sorry I understand it’s ccp4bb forum 
and i am talking about chimera and pymol.
Depending on you need, you can do energy minimization in chimera as well.

Dhiraj


From: CCP4 bulletin board  on behalf of Sam Tang 

Sent: Wednesday, September 29, 2021 6:03 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Add hydrogens

Dear community

This may appear to be a silly question -- I am trying to add hydrogens to the 
structure in PDB 1CDW. My initial thought is to run a single run of refinement 
with a refinement program. It happens that I cannot locate the map coefficients 
under the entry (am I missing something?) So... is there an easy way to do what 
I want in this case?

Warm regards

Sam



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Re: [ccp4bb] [External] [ccp4bb] Protein's C-terminal neutral

[Srivastava, Dhiraj]

Hi Rohit
 Since you are saying the C terminus in crystal structure is not 
end of your protein in crystal, and you are worried about its charge status, is 
in't a good idea to model few residues beyond the C terminus you see in crystal 
structure using weak electron density and crystal packing as guide and then do 
the electrostatic calculation. Even if you are modeling it as COOH, it's not 
going to be true end anyway. Two or three extra residues should be sufficient 
as ionic interaction is going to be quite weak beyond 8-10 A.

Dhiraj

From: CCP4 bulletin board  on behalf of rohit kumar 

Sent: Wednesday, September 22, 2021 4:29 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Protein's C-terminal neutral

Hello Everyone,

Is there any way that I could make my protein's C-terminal neutral using Coot?
Actually, I have a protein-peptide complex structure and my peptide is bound at 
the C-terminal end. While making a surface charge diagram it is negatively 
charged (because of the CO group at the end, which is not the end residue of my 
purified protein)  and I want to make it neutral (possibly a peptide bond in 
solution).

Please let me know if I am clear enough with my question.

Thank you

--
Regards
Dr. Rohit Kumar Singh
Postdoctoral fellow
Aurora CO USA





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Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

[Srivastava, Dhiraj]

Sorry I got confused with the wrong labeling. But the stoichiometry issue and 
poor expression is still the main problem. As Tom suggested, you should try to 
improve the expression. Poor expression often results in impurities 
copurifying. Also, if the interaction is not tight, why are you co-expressing? 
Unless coexpression is helping in some way (solubility ) you are better off 
expressing (and purifying) them separately and then mixing them. often, when 
you co-express multiple proteins, you get poor expression as well.


Dhiraj

From: CCP4 bulletin board  on behalf of Tom Huxford 

Sent: Tuesday, July 13, 2021 11:27 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern.  
Optimize soluble protein expression and pay attention to cell lysis.  Is the 
sample remaining cold throughout the process?  Are you using enough volume of 
buffer relative to solid cell pellet?

Also, from the gel it does not look like your 37 and 15 kDa bands are 
co-eluting.  And your lane labels in the figure are not right.

Good news:  this seems like a solvable problem.  Keep after it!

Tom Huxford.

==
Tom Huxford
Structural Biochemistry Laboratory
Department of Chemistry & Biochemistry
San Diego State University
(619) 594-1606

On Jul 13, 2021, at 5:22 AM, Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:


Hello,

Please find the attached gel picture for the reference and some additional 
information.

Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
cocktail.

SEC column – Superdex 200. 16/300

One of the proteins is 37 kDa while the other one is15 kDa. I do not think that 
they are making that strong heterodimer. The total molecular of the complex 
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower 
temperature (16 ᵒC) but can try around 12 with low IPTG conc.

Regards

Dilip Badgujar

On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
mailto:mahmood.zaig...@gmail.com>> wrote:
Hey Dilip,

There are many reasons for this observation. Rafael is right, please do share 
the image of the gel. Also what are the exact sizes of the two proteins that 
you co-expressed? I have observed the heterodimeric and pentameric proteins on 
SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling 
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can 
perform western blotting, followed by the use of anti-polyhistidine and 
anti-Streptavidin antibodies on separate blots to confirm the suspected bands. 
Likewise SEC followed by WB may confirm the identity of the eluted proteins in 
different fractions after size exclusion chromatography. You may also cleave 
the tags, and then see the magic!

Tom has correctly pointed out that induction at lower temperature is best 
achieved upon incubation of culture so that the temperature is dropped before 
the induction.

Bon Voyage!

-Z


Zaigham M Khan, PhD
Associate Scientist

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States


On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:
Greetings, everyone

I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
Rosetta cells but getting a contaminating band around 60 kDa. One of the 
construct is in pET28a with His tag while the other is in pET21c with Strep tag 
and I am adding all the three selection markers during growth of pre-culture 
and during induction. Initially, cells were grown at 37 °C till OD reaches to 
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do 
purification using Streptactin resin; I can see proteins of my interest bound 
to the resin along with contaminating protein at 60 kDa. I have tried 
performing size exclusion as a follow up step but they are co-eluting in void 
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
with contaminant. I am looking for valuable suggestions to avoid the 
contamination during or after expression.

Thanks in advance.

Regards

Dilip



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--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby




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This message 

Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

[Srivastava, Dhiraj]

Hi Dilip
  It seems like your problem is poor stoichiometry. 37 kda protein 
is poorly expressed. I wouldn’t be worried too much about 60 kda protein. 
At-least at this stage. I might be mistaken but based on previous post, it 
seems like your protein was aggregated. If it is so, you need to improve on 
aggregation and stoichiometry. With better yield of complex, 60 kda impurity 
will be minor.

Dhiraj

From: CCP4 bulletin board  on behalf of Dilip Badgujar 

Sent: Tuesday, July 13, 2021 7:22 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells


Hello,

Please find the attached gel picture for the reference and some additional 
information.

Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
cocktail.

SEC column – Superdex 200. 16/300

One of the proteins is 37 kDa while the other one is15 kDa. I do not think that 
they are making that strong heterodimer. The total molecular of the complex 
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower 
temperature (16 ᵒC) but can try around 12 with low IPTG conc.

Regards

Dilip Badgujar

On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
mailto:mahmood.zaig...@gmail.com>> wrote:
Hey Dilip,

There are many reasons for this observation. Rafael is right, please do share 
the image of the gel. Also what are the exact sizes of the two proteins that 
you co-expressed? I have observed the heterodimeric and pentameric proteins on 
SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling 
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can 
perform western blotting, followed by the use of anti-polyhistidine and 
anti-Streptavidin antibodies on separate blots to confirm the suspected bands. 
Likewise SEC followed by WB may confirm the identity of the eluted proteins in 
different fractions after size exclusion chromatography. You may also cleave 
the tags, and then see the magic!

Tom has correctly pointed out that induction at lower temperature is best 
achieved upon incubation of culture so that the temperature is dropped before 
the induction.

Bon Voyage!

-Z


Zaigham M Khan, PhD
Associate Scientist

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States


On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:
Greetings, everyone

I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
Rosetta cells but getting a contaminating band around 60 kDa. One of the 
construct is in pET28a with His tag while the other is in pET21c with Strep tag 
and I am adding all the three selection markers during growth of pre-culture 
and during induction. Initially, cells were grown at 37 °C till OD reaches to 
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do 
purification using Streptactin resin; I can see proteins of my interest bound 
to the resin along with contaminating protein at 60 kDa. I have tried 
performing size exclusion as a follow up step but they are co-eluting in void 
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
with contaminant. I am looking for valuable suggestions to avoid the 
contamination during or after expression.

Thanks in advance.

Regards

Dilip



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--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby




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[ccp4bb] Eukaryotic protein expression

[Srivastava, Dhiraj]

Hi all
I am trying to express my protein in HEK293 cells for crystallization 
purpose but getting poor expression. I am using cmv promoter. Which 
promoter/vector people use to get good expression in eukaryotic expression 
system?

Thank you

Dhiraj



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Re: [ccp4bb] [External] Re: [ccp4bb] phenix refinement for bent DNA

[Srivastava, Dhiraj]

Thank you everyone for the help. I am sorry about phenix related question. But 
since the question was refinement related I thought it will be ok to ask on 
ccp4bb.

Thank you
Dhiraj

From: Oleg Sobolev 
Sent: Monday, March 29, 2021 6:18 PM
To: Srivastava, Dhiraj 
Cc: CCP4BB@jiscmail.ac.uk 
Subject: [External] Re: [ccp4bb] phenix refinement for bent DNA

Hi Dhiraj,

I have structure with bent DNA. I am trying to refine the structure using 
phenix. do I need to turn off the DNA secondary structure restraints during 
refinement?
Application of secondary structure restraints depends on the quality of the 
experimental data. The most basic parameter to consider would be a resolution. 
For lower-resolution SS restraints might help to keep a reasonable geometry of 
the structure. The bent DNA should also work fine with SS since they are 
restraining base pairs and stacking pairs which normally don't distort too much.

P.S. There is a separate bulletin board for Phenix-specific questions:
http://www.phenix-online.org/mailman/listinfo/phenixbb

Best regards,
Oleg Sobolev.


Thank you
Dhiraj



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[ccp4bb] phenix refinement for bent DNA

[Srivastava, Dhiraj]

Hi
I have structure with bent DNA. I am trying to refine the structure using 
phenix. do I need to turn off the DNA secondary structure restraints during 
refinement?

Thank you
Dhiraj



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Re: [ccp4bb] Cysteine oxidation product

[Srivastava, Dhiraj]

Hi Jon
   there is no surface exposed cysteine nearby which can potentially 
make disulfide bond with the modified cysteine intramolecularily.

Dhiraj

From: Jon Cooper 
Sent: Sunday, November 1, 2020 7:41 AM
To: Srivastava, Dhiraj ; CCP4BB@JISCMAIL.AC.UK 

Subject: Re: [ccp4bb] Cysteine oxidation product

Hello,

does the protein you added cysteine to (I assume you mean by mutagenesis) have 
another cysteine which could make an intra molecular disulphide with it? That 
would probably not show on non-reducing SDS?

Best wishes, Jon Cooper



 Original Message 
On 31 Oct 2020, 15:58, Srivastava, Dhiraj < dhiraj-srivast...@uiowa.edu> wrote:

Hi All
I added an extra cysteine residue to one of my protein interactions 
partners and somehow it increased the affinity in the absence of reducing agent 
however in the presence of reducing agent, the affinity is same. there is no 
disulfide bond formation between the two interaction partner as in non-reducing 
SDS-PAGE, I see the molecular weights corresponding to only the individual 
proteins and not the sum of two.
Does anyone has ever seen this? at pH 8.0, what is the probability that a 
surface exposed cysteine will have a net negative charge and still not very 
reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there any 
such example in literature that anyone knows?

Thank you
Dhiraj



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Re: [ccp4bb] Cysteine oxidation product

[Srivastava, Dhiraj]

Hi Matthias
   I haven’t measured the affinity in a quantitative way. My 
assumption of affinity increase is based on gel filtration. With reducing 
agents, i don’t see comigration of two proteins on gel filtration 
chromatography while without reducing agents, two proteins are co-migrating.

Dhiraj


From: Barone, Matthias 
Sent: Saturday, October 31, 2020 1:29 PM
To: CCP4BB@JISCMAIL.AC.UK ; Srivastava, Dhiraj 

Subject: Re: Cysteine oxidation product


Hi Dhiraj

how did you measure the affinities? What are the two affinities, incl standard 
error (or even amount of data points you used in the titration exp)?

Im just asking because Im curious as what you mean by "increase". If you add 
DDT to an ITC for example, this will create a lot of background signal which 
has to be taken into account. Did you make background titrations for both?



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 Srivastava, 
Dhiraj 
Sent: Saturday, October 31, 2020 4:58:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cysteine oxidation product

Hi All
I added an extra cysteine residue to one of my protein interactions 
partners and somehow it increased the affinity in the absence of reducing agent 
however in the presence of reducing agent, the affinity is same. there is no 
disulfide bond formation between the two interaction partner as in non-reducing 
SDS-PAGE, I see the molecular weights corresponding to only the individual 
proteins and not the sum of two.
Does anyone has ever seen this? at pH 8.0, what is the probability that a 
surface exposed cysteine will have a net negative charge and still not very 
reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there any 
such example in literature that anyone knows?

Thank you
Dhiraj



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[ccp4bb] Cysteine oxidation product

[Srivastava, Dhiraj]

Hi All
I added an extra cysteine residue to one of my protein interactions 
partners and somehow it increased the affinity in the absence of reducing agent 
however in the presence of reducing agent, the affinity is same. there is no 
disulfide bond formation between the two interaction partner as in non-reducing 
SDS-PAGE, I see the molecular weights corresponding to only the individual 
proteins and not the sum of two.
Does anyone has ever seen this? at pH 8.0, what is the probability that a 
surface exposed cysteine will have a net negative charge and still not very 
reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there any 
such example in literature that anyone knows?

Thank you
Dhiraj



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Re: [ccp4bb] tetrameric tag for extracellular proteins

[Srivastava, Dhiraj]

P53 tetramerization domain might be a way to go.


From: CCP4 bulletin board  on behalf of Comolettis 
<85822939...@gmail.com>
Sent: Friday, September 25, 2020 9:59 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] tetrameric tag for extracellular proteins

Hi all,

Following what someone recently suggested, Fc tag is a great way to dimerize a 
secreted protein. I have successfully tried the TNF receptor-associated factor 
1 tail to trimerize and the COMP (cartilage oligomeric matrix protein) to 
pentamerize my monomer but I can't identify a suitable tag to tetramerize my 
construct.
Any pointer?

Thanks,
Davide Comoletti
VUW, Wellington, NZ



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Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Srivastava, Dhiraj]

Thanks everyone for all the nice suggestions.
regarding Matthew's question, Yes, There is a possibility of polydispersity due 
to the proteins making chain/aggregate, however the affinity between the 
dimeric protein A and monomeric protein B is poor and kinetics of dissociation 
is very fast thus complex can not survive gel filtration chromatography. So, by 
increasing the avidity, We are hoping that only 2:2 complex will survive the 
gel filtration and we will be able to isolate monodispersed complex for further 
biophysical studies like SAXS or crystallization.

Thank you
Dhiraj


From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Tuesday, September 22, 2020 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

I was thinking the form of GFP that dimerizes.  This would also make it easy to 
track where the protein is.

On Tue, Sep 22, 2020 at 1:28 PM Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>> 
wrote:
Any dimeric tag should work if you add a long enough linker to satisfy your 
distance criterion.

GST, for example. Download the coordinates and get a rough idea how long the 
linker would have to be for your protein.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj 
mailto:dhiraj-srivast...@uiowa.edu>> wrote:


EXTERNAL MAIL

Hi
I want to make my protein dimeric to increase its affinity for its 
interaction partner which is a dimer. does anyone know a suitable tag/fusion 
protein which can be used as C terminal fusion for this purpose? I can not use 
any of the leucine zipper as I am looking for the distance between the c 
terminus to be around 30-40 A.


Thank you
Dhiraj


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or attachments, and validate the sender's email address before replying.




UT Southwestern

Medical Center

The future of medicine, today.



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[ccp4bb] dimeric tag to induce the homodimerization of protein

[Srivastava, Dhiraj]

Hi
I want to make my protein dimeric to increase its affinity for its 
interaction partner which is a dimer. does anyone know a suitable tag/fusion 
protein which can be used as C terminal fusion for this purpose? I can not use 
any of the leucine zipper as I am looking for the distance between the c 
terminus to be around 30-40 A.


Thank you
Dhiraj



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Re: [ccp4bb] number of frames to get a full dataset?

[Srivastava, Dhiraj]

Well, its not no. of frames but minimum degree of crystal rotation. A goggle 
search gave me this article.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557013/


Dhiraj

From: CCP4 bulletin board  on behalf of Murpholino 
Peligro 
Sent: Monday, June 22, 2020 5:03 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] number of frames to get a full dataset?

Hi.
Quick question...
I have seen *somewhere* that to get a 'full dataset we need to collect n 
frames':
at least 180 frames if symmetry is X
at least 90 frames if symmetry is Y
at least 45 frames if symmetry is Z
Can somebody point where is *somewhere*?

...also...
what other factors can change n... besides symmetry and radiation damage?

Thanks



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Srivastava, Dhiraj]

As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Srivastava, Dhiraj]

As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Changes in quaternary structure

[Srivastava, Dhiraj]

Pyruvate kinase specially M2 isoform is one example which exists in tetramer 
and dimer/monomer state. Several ligands affect its shape and oligomeric state, 
thus affecting its activity.

Dhiraj

From: CCP4 bulletin board  on behalf of Gabriela GARCIA 
RODRIGUEZ 
Sent: Wednesday, October 9, 2019 6:14 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Changes in quaternary structure

Dear CCP4BB subscribers,

I am writing a discussion on extensive quaternary changes between two different 
homodimers of the same protein and I would like to ask you for any examples of 
your own work/that you know of to include in my analysis. Basically, any 
proteins known to exist in two different (or more) homodimerised states 
(suffering changes in domain organisation between states would be even more 
interesting).
I would greatly appreciate any examples! Thank you for your time and attention.

Warm regards,

Gabriela


Gabriela Garcia Rodriguez

gabriela.garcia.rodrig...@vub.be

PhD candidate

Structural Biology Research Centre (SBRC)

Vlaams Instituut voor Biotechnologie (VIB) - Vrije Universiteit Brussel (VUB)

Pleinlaan 2- 1050 Brussels, Belgium








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Re: [ccp4bb] Only C-terminal GFP-tag of fusion protein expression in Pichia Pastoris.

[Srivastava, Dhiraj]

I am not much aware of pichia system but in general, its quite possible your 
protein is getting protelysed and all that’s leftover is gfp. Although I expect 
mbp to be there as well as its quite well behaved tag. You might want to 
analyze whole cell lysate to see if your protein is there. A time point 
analysis for expression might be helpful. You might want to express it for less 
time.

Dhiraj

From: CCP4 bulletin board  on behalf of dongxiaofei 
<18811070...@163.com>
Sent: Friday, October 26, 2018 12:40:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Only C-terminal GFP-tag of fusion protein expression in 
Pichia Pastoris.


Dear All,

 I'm focusing on expressing my fusion protein in Pichia pastoris. The 
size of my protein is about more than 200 kDa, and there are mbp-tag and 
GFP-his-tag at N-terminus and C-terminus. Verify that my pPICZb plasmid  is no 
problem by sequencing.

 After  centrifugation, load  the supernatant to amylose  column and NI column, 
only NI beads have green fluorescence. No strips in the eluent of amylose, and  
a strip of gfp-tag for NI column in SDS-PAGE.

Only C-terminal GFP-tag of my fusion protien was expressed !

Why  pichia pastoris  expresses C-terminal GFP-tag, but neglect my protein 
sequence?  What should I do to solve this problem and get my protein in Pichia 
Pastoris?



Would be very grateful for any advice!

Thanks,

Dong Xiao







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[ccp4bb] crysol or FoxS integration into python and pymol

[Srivastava, Dhiraj]

Hi All

 sorry for asking non-crystallography related question. Is it possible 
to integrate crysol or FoxS in python and pymol? I have generated thousands of 
models of my protein that I want to test against SAXS and other biochemical 
data. The ability to integrate the comparison of experimental and calculated 
X-ray scattering profile into python and pymol will be really helpful as I can 
easily calculate other parameter of my protein in pymol and compare it to other 
biochemical data. There is a pymol plugin available for SAXS but its not 
helpful for me as its not practical to use plugins for so many different 
models. I am looking for script that can automate the calculation.

 if anyone has script that can do this and is willing to share with 
me, that will be really helpful.


thank you

Dhiraj



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Re: [ccp4bb] sodium thiocyanate as cryoprotectant

[Srivastava, Dhiraj]

Probably you can not use thiocyanate as cryoprotectant because its a chaotropic 
agent. Although its a very mild denaturant (destabilizer) but at high 
concentration, it can destroy your crystals. I have recently used 30 % sucrose 
for cryo protection of crystals growing under same condition and it worked 
beautifully. My crystals were cracking in glycerol.

Dhiraj

On Sep 27, 2016, at 5:43 PM, Alex Lee 
> wrote:

Hi CCP4bb members,

I have a condition for my protein crystals at "0.2M sodium thiocyanate, 25% 
PEG3350, pH6.9".

I'd like to try cryoprotectant with just higher concentration of sodium 
thiocyanate, but I have no google hits of the range of concentration I should 
use.

I wonder anyone in this forum tried to use sodium thiocyanate as 
cryoprotectant?  What's the concentration range for this if used as 
cryoprotectant?

Thanks.

[ccp4bb] different Kd and Km value

[Srivastava, Dhiraj]

Hi
   I have a protein with two substrate. when I am doing the binding studies 
with the two substrate separately, I am finding one of the substrate to have 
similar kd and Km. however the km and kd values are almost 30 fold different 
for the other substrate. it binds 30 fold more tightly then you can think based 
on Km. I don't know how to explain this. Km and Kd can be different but does 
any one have seen that much difference in Kd and Km?


Thank you
Dhiraj

Re: [ccp4bb] phosphoprotein crystallization

[Srivastava, Dhiraj]

Thank you Prof Lewis.
Its serine residue which is getting phosphorylated. the pI of my protein is 
around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried 
ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow 
through or very poorly binding to Q and SP sepharose. does any one have any 
good suggestion for finding out phosphorylation status of a basic protein with 
pI around 8.0. So far we were using mass spec on tryptic digest but I need a 
simple and relatively quick method because its not practical to run mass spec 
on each and every fractions before pooling them. 

Thanks
Dhiraj
On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote:

 is this a phosphorylation on serine, threonine or tyrosine? if so, they 
 should be quite stable between ~pH 5 and 8. the MS results are probably not 
 quantitative, just qualitative, and i would expect that the addition of a net 
 negative 2 charge from the phosphoryl group should make a difference to your 
 protein on ion exchange.
 
 i would run a non-denaturing PAGE, and look for differences in Rf for your 
 protein. i bet that the phosphorylation has not gone anywhere near 
 completion, especially since you seem to expect a conformational change to 
 occur to accommodate the PO3, and this is not observed. if your preps are 
 clean, you shouldn't need a p'tase inhibitor.
 
 good luck
 
 rick
 
 On 19/01/15 16:54, Dhiraj Srivastava wrote:
 I am trying to crystallize a protein that I am phosphorylating in-vitro. 
 There is no way that I can purify the phosphorylated protein from 
 unphosphorylated one. I tried Ion exchange and gel filtration for separating 
 them and they are not working.
 Mass spec result shows the phosphorylation at desired site but in the 
 crystal structure, I am not seeing density for phosphoryl group. I haven't 
 use any phosphatase inhibitor during crystallization. is it possible that 
 phosphoryl group is getting lost due to radiation damage or due to 
 phosphatases, my protein is getting dephosphorylated before making crystal? 
 I typically get crystals within 24 hours. if I am losing phosphoryl group 
 due to radiation damage, should I still expect to see conformational changes 
 due to phosphorylation? in phosphorylated protein, an alpha helix has to 
 move to accommodate phosphoryl group but I am not seeing any change in that 
 alpha helix. did any one tried to set crystal tray with phosphorylation 
 reaction mix without further purification?
 
 thank you for suggestion.
 
 Dhiraj
 
 -- 
 R. J. Lewis
 Professor of Structural Biology
 Institute for Cell and Molecular Biosciences
 Faculty of Medical Sciences   Tel: +44 (0)191 208 5482
 University of Newcastle   Fax: +44 (0)191 208 7424
 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk
 URL: sbl.ncl.ac.uk
 

Re: [ccp4bb] off-topic: tag removal

[Srivastava, Dhiraj (MU-Student)]

See the following reference-

  Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 
Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing 
Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Philipp Ellinger 
[filu...@gmx.de]
Sent: Wednesday, February 23, 2011 3:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: tag removal

Dear all,

I have a question concerning removal of a his-tag sequence.
We have crystallized a protein with an important feature at the C-terminal part 
of the protein.
Unfortunately, we cannot express it with a N-terminal his-tag, only with a 
C-terminal his-tag.

Therefore we are looking for a protease which cleaves off the sequence without 
leaving any extra amino acid on the C-terminus of our protein. Meaning we 
obtain really the wild type protein.
Does anyone know about a protease or cleavage site which is completely removed?


Many thanks in advance

Phil

[ccp4bb] per-residue RMSD calculation for homologous structure

[Srivastava, Dhiraj (MU-Student)]

Hi All
   does anyone know any software that can calculate and print out RMSD of 
every residue (c alpha will be good) for homologous structures which has only 
30-40 % sequence similarity? I looked on the web but all the software that I 
found require the sequence to be the same for both structure.

Thank you

Dhiraj