Re: [ccp4bb] Name that protein!

[Vivoli, Mirella]

It is very similar to a voltage-gated potassium or sodium channel.
Beautiful structure!

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From: CCP4 bulletin board  on behalf of Sorin Draga 

Sent: Wednesday, June 27, 2018 1:41:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Name that protein!

the closest thing I can think of is tetramer of hAQP4

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678640/


On Wed, Jun 27, 2018 at 2:07 PM, david lawson (JIC) 
mailto:david.law...@jic.ac.uk>> wrote:
Hi All

Apologies for the off-topic question.

The attached image has been hanging on the wall at our institute for several 
years and nobody knows what it is or who generated it.

If anyone recognises it, please let me know.

Many thanks

Dave Lawson

---

Dr. David M. Lawson
Department of Biological Chemistry,
John Innes Centre,
Norwich,
NR4 7UH, UK.
Tel: +44-(0)1603-450725
Fax: +44-(0)1603-450018
Email: david.law...@jic.ac.uk




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Re: [ccp4bb] Help with assigning density

[Vivoli, Mirella]

Hi Tristan!
Definitely PEG, in the so called horseshoe shape.
Cheers,
Mirella

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From: CCP4 bulletin board  on behalf of Tristan Croll 

Sent: Friday, January 26, 2018 6:09:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Help with assigning density

Yes it can. See 5x49 (residue 603) for a wonderful example.

On 2018-01-26 16:46, Michal Boniecki wrote:
> I have positive density around lysine residue (image). This density is
> found in all crystals, and only with this lysine. It is solvent
> accessible but again not only this one is surface K residue. Can PEG
> wrap around lysine like this? Maybe someone else have seen similar
> density and knows what it might be?
>
> Thank You

Re: [ccp4bb] Predict effects of mutations on protein stability and protein-protein interaction

[Vivoli, Mirella]

Dear Wenhe,
I have used iMutant 2.0.
I hope it helps.
Below the link of a paper related to the prediction server:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1160136/

Best,

Mirella

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From: CCP4 bulletin board  on behalf of WENHE ZHONG 

Sent: Friday, November 24, 2017 6:08:28 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Predict effects of mutations on protein stability and 
protein-protein interaction

Dear Community,

A little bit out-of-topic here. We have a few interesting sites would like to 
mutate on proteins to test the protein stability and improve the 
protein-protein interactions. Before moving forward to the site mutagenesis 
experiment, we like to do some prediction first to narrow down the candidates. 
We only know that SDM is good server to predict the protein stability. Do you 
have other servers or softwares can recommend? We can do a cross comparison.

Thanks.

Kind regards,
Wenhe

Re: [ccp4bb] off topic

[Vivoli, Mirella]

Hi Anamika,
I guess you tried several growth conditions.
You might try to do a heat shock growth.
Grow at 37C until OD 0.6, then move the bacteria to 42C for half hour, then to 
30C and grow them for 20 hours.
Another strategy is to add just before induction 1,2 or 3% Ethanol.
Not sure these will work with your protein but you might give a go.
Other strategies I could suggest is to clone STAT1 protein with other tags 
(MBP, GST, and so on) or in othe expression system, like ArticExpress.
If none of them works, then there is the painful strategy of refolding.
Feel free to contact me, I would be happy to help you.
Cheers,

Mirella

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From: David Blum
Sent: Thursday, 6 July, 22:10
Subject: Re: [ccp4bb] off topic
To: ccp4bb@jiscmail.ac.uk


Hi Anamika,


I did a search and it looks like researchers are using either mammalian cells 
or baculovirus to express this protein.  I don't have experience with this 
particular construct so could you tell me why you choose E. coli?  I run a 
protein expression facility and we typically use HEK or CHO cells and get mg 
amounts from cell culture of mammalian derived proteins like STAT1.  Happy to 
talk offline if that would be easier.


Best,

David

--

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

(706) 542-1035 (Office)



On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh 
> wrote:

Hi,


Is anyone has worked with STAT1 proteins?


I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was 
no expression so far or rather say inconsistent expression. Sometimes the 
expression was in inclusion bodies.  I have tried different methods to pull out 
the protein from inclusion bodies using urea, guanidium chloride tween20 but 
none of them worked well. The yield was very low (very faint band on SDS-PAGE ) 
from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells 
but no success so far.


We thought to use some other vector system like with SUMO tag but did not 
proceed because the aim of the project to design inhibitor and tag will 
interfere.


Please suggest me something so that I can complete my project in lesser time.



Looking forward to valuable suggestions.


Thanks

Anamika

Re: [ccp4bb] Possibility of Quasi-crystal formation of heterodimeric protein complex

[Vivoli, Mirella]

Dear Debiasish,
it looks like you got spherulites.
You might try to optimize the conditions or use these spherulites for 
microseeding. It worked for me once.
Good luck!

Mirella

Sent from my iPhone

On 24 Mar 2017, at 08:29, Camillo Rosano 
> wrote:

Dear Debiasish,
   more than "quasicrystals" (crystal in which molecules 
adopt an ordered but aperiodic structure), to me the one in your photo looks 
like spherulites. You shouldn't be far for finding the right crystallization 
conditions: maybe change the pH of your precipitant and try lower concentration 
of protein or precipitant or both at the same time.
Camillo

On Fri, Mar 24, 2017 at 12:10 AM, Debasish Kumar Ghosh 
> wrote:
Dear All,

I have a small doubt regarding possibility of formation of quasi-crystal of 
protein complex. I am trying to co-crystallize a heterodimeric protein complex 
of two small proteins (14KDa and 16KDa, both are human origin). One of them is 
a membrane protein. we have extensively characterized their qualitative and 
quantitative binding properties (with IP/IB, Confocal microscopy, ITC etc.). We 
are quite certain of their strong 1:1 binding stochiometry.
In some crystallizing conditions, we are getting some structures which appears 
to me as quasi-crystals (Image attached). However, I am not coming across ample 
examples of quasi-crystals of protein complexes.
I will be very keen to know if anyone had this (similar) kind of experience(s). 
And of course it would be wonderful to know if they are diffractable and, if 
yes, what are the odds of having good diffraction.


Best !!

Debasish

CSIR- Senior Research Fellow
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Re: [ccp4bb] How to determine the concentration of biotinylated peptide?

[Vivoli, Mirella]

Hi Alex,
you can measure the absorbance at 214-220 nm, which is where the peptide bonds 
absorb, but you should know/calculate/predict the extinction coefficient of 
your peptide at that wavelength. 
Furthermore, you might try BCA assay which is colorimetric as the bradford but 
the reaction involves the peptide bonds (it has low sensitivity but an increase 
in temperature during the assay might help).
Densitometry would be another way but but is give you only a rough idea: I 
would not recommend to do it.
Finally, if you have
the possibility to do it, the aminoacid analysis represents the golden standard 
analysis for a precise quantitation of peptides: this method involves a 
hydrolysis step, a separation by HPLC, detection.

Cheers,
Mirella








Sent from my iPhone

> On 5 Feb 2017, at 22:33, Alex Lee  wrote:
> 
> Dear All,
> 
> Sorry for the off-topic question, I'd like to do Biacore SPR assay with 
> N-terminal biotinylated peptide as ligand (to Biacore SA chip) and my protein 
> as analyte. I have a question of how to determine the concentration of 
> biotinylated peptide （synthetic peptide), if the peptide has no Tyr and no 
> Trp residue, I guess amino acid analysis may not work because the N-terminal 
> of the peptide is biotinylated. 
> 
> I'd appreciate if anyone share his/her experience on this.
> 
> 

Re: [ccp4bb] Unknown electron density blob

[Vivoli, Mirella]

Hi there,

I think it is PEG, very common to have PEG in horseshoe shape with arginine 
pointing toward to it.

Cheers,


Mirella


Vivoli Mirella

Associate Research Fellow
Biocatalysis Centre, Henry Wellcome Building
College of Life and Environmental Sciences,
University of Exeter
Stocker Road
Exeter
Ex4 4QD
Tel: + 44 (0)1392 726121
Email: m.viv...@exeter.ac.uk

"I don't want to believe. I want to know". [C. Sagan]

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Paul Emsley 
<pems...@mrc-lmb.cam.ac.uk>
Sent: 24 January 2017 17:32:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Unknown electron density blob

On 24/01/2017 17:19, Uma Gabale wrote:

> While refining a structure at 2.5 A resolution, we observed a 
> semi-circular/crescent shaped
> electron density blob as shown in the attached picture. We have been unable 
> to identify it
> so far, and would appreciate any help in identification.

If you have Coot:

Extensions->Modelling->Add Other Solvent Molecules->Add New Residue 
Type->"PG4"->Add
then click "PG4 Tetraethylene Glycol"

Re: [ccp4bb] off-topic: protein losing FAD during purification

[Vivoli, Mirella]

Dr. Stefano,
1) I would add FAD in all buffers after lysis,even after GF you could perform 
dialysis with buffer supplemented with FAD.
2)for the second question I found this paper which could be helpful:
Large-scale preparation and reconstitution of apo-flavoproteins with special 
reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. 
Hydrophobic-interaction chromatography.
Good luck,
Mirella

Sent from my iPhone

On 13 Mar 2014, at 22:40, Benini Stefano (P) 
stefano.ben...@unibz.itmailto:stefano.ben...@unibz.it wrote:

Dear All (those dealing with wetlab stuff..),

While purifying a FAD containing protein we lose part of the FAD (on the gel 
filtration we clearly see two bands corresponding to holoprotein and free FAD).

We obtain crystals but diffracting to only about 4 A despite their beautiful 
look. Our hypothesis is that the crystals contain a population of molecules 
with and without FAD (?).

The questions are:

1) how to keep FAD bound to the protein during purification and crystallization?

2) how to completely remove FAD from the protein?

Thank you very much for any help provided!

Best regards

Stefano (part-time wetlab person)


Dr Stefano Benini, Ph.D.
Assistant Professor

First International workshop: Molecular Basis of Fire Blight, Bolzano 
15.10.2014

Laboratory homepage:
http://pro.unibz.it/staff2/sbenini/B2Cl.htm

Personal homepage
http://pro.unibz.it/staff2/sbenini/

I don't like anything that's fake and I hate pretenders!

*
Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl)
Faculty of Science and Technology
Free University of Bolzano
Piazza Università, 5
39100 Bolzano, Italy
Office (room K2.14):  +39 0471 017128
Laboratory (room E.021): +39 0471 017910
Fax: +39 0471 017009

ogni giorno in più è un giorno in meno.

[ccp4bb]

[Vivoli, Mirella]

Dear Prerana,

before starting the crystallization you could try to check the state of you 
protein, simply determining the Aggregation Index (AI) from measuring the 
absorbance at 280 nm and 340 nm. The AI is then computed using this simple 
formula: AI= 100 x (Abs340/(Abs 280 - Abs 340)). Soluble and non-aggregated 
proteins solutions typically have an Aggregation Index of 2 and lower, whereas 
for some aggregation will be 2-5;  heavily aggregated proteins show an 
aggregation index 5. Of course you cannot use Nanodrop for it (because the 
wavelength for the baseline normalization is 340 nm). I would try to decrease 
the concentration of your protein to 4-5 mg/ml.Good luck.

Best,

Mirella


Vivoli Mirella

Postdoctoral Fellow
University of Exeter,
Biosciences
Biocatalysis Centre, Henry Wellcome Building
Stocker Road,
Exeter,
Ex4 4QD
Tel:+ 44 (0)1392 726121



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. 
[tracy...@gmail.com]
Sent: Friday, February 21, 2014 11:14 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb]

Hi, Sorry for asking an off-topic question,
I have recently purified a protein having a molecular weight of 40kDa and 
concentration of the protein was 8mg/ml.  When I tried to set the protein for 
crystallisation using micobatch method, the protein started precipitating in 
most of the buffer conditions of Crystal screen and Peg ion. The precipitation 
took place very quickly (within 5-10 mins).
How should I overcome this problem?


Regards
Prerana

Re: [ccp4bb] problems with cleaving protein

[Vivoli, Mirella]

Dear Kris,

the release of the desired protein from a fusion protein is affected by the 
adjacent amino acid sequences (e.g. the presence of a Proline after Lys makes 
the protease activity unworkable)  at the cleavage site as well as by the size 
of the two fused components and of course by the accessibility of the cleavage 
site (the protein folding could bury the cleavage site). Moreover, did you try 
different enzyme/substrate ratio and different time of incubation to find out 
suitable conditions? Another thing I know is that high concentration of salt 
such as NaCl inhibits the cleavage reaction, as well the presence of unsoluble 
aggregates. I would suggest to perform a dialysis step to remove any salt if 
you have and dilute the fusion protein to 0.2-0.5 mg/ml. Which kind of buffer 
are you using in the purification process? Phosphate buffer reduces the 
activity of the enzyme. Finally if it doesn't work changing these conditions, I 
would suggest to use a proper concentration of denaturing agents (low 
concentration) to slacken the fusion protein structure and to increase then the 
accessibility of the cleavage site (if it is the case of a buried cleavage 
site).

Best,

Mirella

Vivoli Mirella

Postdoctoral Fellow
University of Exeter,
Biosciences
Biocatalysis Centre, Henry Wellcome Building
Stocker Road,
Exeter,
Ex4 4QD
Tel:+ 44 (0)1392 726121



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of K Singh 
[ksc...@gmail.com]
Sent: Sunday, July 21, 2013 8:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] problems with cleaving protein

Dear All,
I have expressed fusion protein that is expressed in the following format

Fusion partner - Enterokinase cleavage site - Protein of interest.

somehow enterokinase is not doing its job though following manufacturers 
recommendations. any inputs like what protocol worked.

Many thanks for your inputs

Kris