Re: [ccp4bb] Suggestions on Dynamic Light Scattering instruments

[Debasish Kumar Ghosh]

Dear Chandramohan,

We use Malvern particle size analyzer (ZEN 3690 ZETASIZER NANOZS 90, version 
7.03) fitted with 21CFR part 11 software. It works fine for us in terms of 
measuring soluble proteins particles in size range from nanometre to micrometre.

Hope this helps.

Cheers!!

Debasish

__

Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Chandramohan Kattamuri <1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, 30 Apr 2018 20:58:33 +0530 (IST)
Subject: [ccp4bb] Suggestions on Dynamic Light Scattering instruments

Dear all,

We are looking for Dynamic Light Scattering instruments to test our purified 
protein complexes prior to crystallization. Any suggestions or recommendations 
regarding which instruments to pursue?

Thanks in advance


Chandra

kattamuricha...@yahoo.com

Re: [ccp4bb] domain prediction of a membrane protein

[Debasish Kumar Ghosh]

Hi Firdous,

The question, if I am understanding correctly, is to find the topology of the 
regions of a membrane protein, that is cytoplasmic, membranous or 
extra-cellular. You can do it by site directed cystine mutagenesis nearby to 
the the preferred region/domain followed by MAL-PEG analysis. There are several 
studies using this approach to find the topology of membrane protein. 
You must put a tag like HA or 3XFLAG nearby to the domain/region.
Regarding the difficulties in expressing membrane protein, they are usually 
toxic for cells in over-expressed condition. So, a careful approach would be 
growing bacterial culture at lower temperature like 18 degree celcius and also 
putting lower concentration of IPTG (0.1mM) as the final concentration in 
culture (if it's a T7 based expression system). 
Hope this helps.

Best!!

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Firdous Tarique 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, 11 Dec 2017 08:03:37 +0530 (IST)
Subject: [ccp4bb] domain prediction of a membrane protein

Hello everyone

I am trying to clone some domains of a transmembrane protein. I have used
an online server "Phobius" for the prediction of sequences which is showing
it to be a membrane protein with cytoplasmic, non cytoplasmic and
transmembrane regions. Attached is the image of that prediction. Regions
from 100-200 amino acid residues which I am more concerned about is
predicted to be cytoplasmic in nature while other servers have predicted it
to be non cytoplasmic in nature. I am having problems in expressing this
domain. Sometimes the prediction may go wrong for such a short stretch of
amino acid residues.

My question is to know about any technique or any classical experiment
which can actually tell me the exact location of this domain across the
membrane. I mean real experiments not predictions.

Regards

Firdous
Graduate student
Delhi University
India

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

[Debasish Kumar Ghosh]

Hi Wenhe , 

You can mutate the desired residue in 'pymol' (save the new pdb structures) and 
run them for molecular dynamic simulations (in Gromacs, Desmond etc.) to see 
the stability of your protein [for the full length protein or its segments] (by 
checking RMSD and RMSF values). You can analyze various aspects using the 
trajectory files and ensemble average values. 
Hope this helps. 

Best!! 

Debasish 

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

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com 
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) 
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html 



- Original Message -

From: "WENHE ZHONG"  
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Friday, November 24, 2017 10:38:28 PM 
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] Precipitation issue during refolding

[Debasish Kumar Ghosh]

Dear Dipankar, 

Your problem is quite tricky to solve. I have two opinions which worked for me 
for a very high aggregation prone protein (Huntingtin, which always used to go 
in inclusion bodies and precipitate even in elution buffer). 
1. First have 4-5M Guanidinum Hydrochloride in the lysis buffer during the 
purification process, and keep 150mM proline in the elution buffer. I have 
experienced that addition of proline greatly reduces the aggregation 
propensity. 
2. Second option is dealing the problem with advantage of that problem. Try to 
precipitate the protein (which is in elution buffer) with acetone (soon after 
elution) and resolubilize it in your desired final buffer very rapidly under 
cooling conditions (like in 4 degree centigrade). Though the 100% protein may 
not resolubilize, but it will fairly solubilize as maximum as up to 90% (as 
that happened with me). My intuition is that in the final solubilized fraction 
you will get high proportion of folded form of your protein. 
Just check if it works for you. 

Best wishes!! 

Debasish 

CSIR- Senior Research Fellow 
C/o: Dr. Akash Ranjan 
Computational and Functional Genomics Group 
Centre for DNA Fingerprinting and Diagnostics 
Hyderabad, INDIA 

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com 
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) 
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html 



- Original Message -

From: "Dipankar Manna"  
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Thursday, September 21, 2017 3:42:28 PM 
Subject: [ccp4bb] Precipitation issue during refolding 

Hi, 

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with. 
​ 
Any suggestion to deal this issue would be highly appreciated. 

Thank you in advance. 

Best, 

Dipankar​ 

-- 
Dipankar Manna, Ph.D 
Postdoctoral Researcher 
Department of Molecular Medicine 
Institute of Basic Medical Sciences 
University of Oslo, Domus Medica 
Oslo, Norway 

Mob : +47 451 66 517 
E-mail: dipankar.ma...@medisin.uio.no 
dipankar.biot...@gmail.com 
http://www.med.uio.no/imb/english/people/aca/dipankam/ 

Re: [ccp4bb] Hydrophobic hotspots

[Debasish Kumar Ghosh]

Hi Hugh,

Waltz is an excellent web-server to give very good results on amyloidogenic 
regions based on sequence stretches (correlate the regions with hydrophobic 
patches by hydropathy plot obtained from Expassy-Protscale). Aggrescan and 
PASTA are two other reliable servers for the same kind of prediction (though I 
have more preference for Waltz).
Based on the 3D structure (pdb structures) you can use the Maestro program 
(from Schrodinger Inc.) to get the aggregation prone surface(s) / aggregation 
prone surface regions.  

Best!!

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "Hugh Morgan" <1103e90ebb53-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, July 26, 2017 10:35:07 PM
Subject: [ccp4bb] Hydrophobic hotspots

Hi all, can anyone recommend a program for identifying hydrophobic 
(aggregation) hotspots using either the amino acid sequence and/or structural 
data. 
Thanks in advance for your help
Hugh

Ps. Have tried aggrescan but would like to try a few others and compare, 
ideally a more structural based program.

Re: [ccp4bb] Primer design

[Debasish Kumar Ghosh]

No need of the whole exome. Sequencing The second PCR product will do the job I 
guess. Second PCR (from the cDNA pool) with specific forward primer and and 
oligodA reverse primer. Surely a matter of less than $3 

Best,

DKG


- Original Message -
From: "Jacob Keller" <kell...@janelia.hhmi.org>
To: "Debasish Kumar Ghosh" <dkgh...@cdfd.org.in>, CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 6:48:25 PM
Subject: RE: Primer design

Or sequence the whole exome for what, $500-1000?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish 
Kumar Ghosh
Sent: Monday, July 24, 2017 7:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Primer design

Dear Syed,

The process is very trivial to clone your gene of interest. Assuming your gene 
is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse 
primer. First isolate the total RNA from the tissue or cells and do the cDNA 
synthesis using oligodT primer followed by gene specific PCR with forward 
primer and oligodA primer from the cDNA pool. Sequence the PCR product to get 
to know the first stop codon in the ORF. Making the specific reverse primer 
from the sequence is then just matter of time.  
Hope this helps.

Best wishes,

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group Centre for DNA Fingerprinting and 
Diagnostics Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: 
http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 4:56:00 PM
Subject: [ccp4bb] Primer design

Hello All

I am interested in cloning a gene from a plant. I searched the database only 
partial sequence is available, ie: for 160 residues only. The full length of 
the protein is around 570 residues. I designed forward primer and I have no 
clue to design reverse primer.

Any help

Thank you

Syed

Re: [ccp4bb] Primer design

[Debasish Kumar Ghosh]

Dear Syed,

The process is very trivial to clone your gene of interest. Assuming your gene 
is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse 
primer. First isolate the total RNA from the tissue or cells and do the cDNA 
synthesis using oligodT primer followed by gene specific PCR with forward 
primer and oligodT primer from the cDNA pool. Sequence the PCR product to get 
to know the first stop codon in the ORF. Making the specific reverse primer 
from the sequence is then just matter of time.  
Hope this helps.

Best wishes,

Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, July 24, 2017 4:56:00 PM
Subject: [ccp4bb] Primer design

Hello All

I am interested in cloning a gene from a plant. I searched the database only 
partial sequence is available, ie: for 160 residues only. The full length of 
the protein is around 570 residues. I designed forward primer and I have no 
clue to design reverse primer.

Any help

Thank you

Syed

Re: [ccp4bb] Separating Monomers and Dimers

[Debasish Kumar Ghosh]

Dear Jaimohan,

With high amount of purified protein (like obtained in recombinant expression) 
they are likely to form higher oligomers if they have intrinsic property of 
association. With time the content of oligomers do increase. In SDS-PAGE even, 
you will see a faint band of oligomer. This is due to higher mass of protein 
from which some still could form oligomer even after boiling and presence of 
SDS. I am sure your protein has higher content of helical fraction.
My suggestion to you would be to perform HPLC or FPLC every time before assay 
or exp. It would reliably separate the monomer and higher oligomers. 

Best regards,

Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: jai mohan <0cab66323371-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tue, 27 Jun 2017 17:52:34 +0530 (IST)
Subject: [ccp4bb] Separating Monomers and Dimers

Dear all,I am working on a Red FluorescentProtein (His-Tag) molecular weight 
around 27kDa. After purification I ran a SDSpage, the band at 27kDa confirms 
the monomer. The protein was stored at -20C, aweek later again I ran a gel, 
this time I saw another new band between 50-60kDa, it confirmsthe protein 
solution contains both monomers and dimers. I would like to know, whatis the 
best way to separate the monomers and dimers? One of my colleague adviceme to 
go for sucrose gradient centrifugation and size exclusion 
chromatography.However, I seek all your valuable suggestions and advice.
With best regardsDr. S.M.Jaimohan

[ccp4bb] Spam : Re: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

[Debasish Kumar Ghosh]

Assuming the protein is in solution, drop cast 2ul of solution onto a glass 
surface and spread evenly with a sharp needle. Air dry in dust free chamber and 
blow the surface with gentle stream of nitrogen gas. Proteins stick to surface 
quite well to be analyzed for various microscopy (AFM, SEM etc.). If the 
purpose is not to do any surface probe microscopy, coating the glass surface 
with Sigmacoat helps in better binding. Poly-lysine coated glass cover slips 
can be alternative but mostly not preferred for small proteins or peptides.

Hope that helps.

Best!!


Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Jacob Keller 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 05 Apr 2017 08:49:40 +0530 (IST)
Subject: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

Does anyone have a simple way to attach purified his-tagged protein solidly to 
a coverslip?

Thanks,

Jacob Keller

***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

[ccp4bb] Spam : Re: [ccp4bb] Protein Ligand interaction using SPR technique

[Debasish Kumar Ghosh]

Dear Praveen,

We have very good experience with protein ligand interaction with Biacore 3000 
using CM5 chip. The NTA chip is prone to nonspecific interactions and give 
erroneous results. However the gold standard is ITC since it is more sensitive 
than Biacore 3000. 
One quick note for using Biacore 3000 is to optimize the buffer. HBS appeared 
nice for us.
Hope that helps.

Cheers!!



Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Praveen Tripathi 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, 31 Mar 2017 10:31:43 +0530 (IST)
Subject: [ccp4bb] Protein Ligand interaction using SPR technique

Dear all,
Sorry for off-topic question.
I want to study protein interaction with few small molecules using SPR
(Machine- *BIACORE 3000*).

The recombinant protein expressed in bacterial expression system is of 92
kDa (with His tag), pI= 9.

*Question-*
1. What should be the chip of choice- *NTA chip or CM5 chip* of GE
healthcare?
2. Is there any alternative available for chips and machine?

Thanks in advance.

Regards

Praveen

[ccp4bb] Spam : Re: [ccp4bb] Protein Ligand interaction using SPR technique

[Debasish Kumar Ghosh]

Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Praveen Tripathi <tripathipraveen2...@gmail.com>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, 31 Mar 2017 10:31:43 +0530 (IST)
Subject: [ccp4bb] Protein Ligand interaction using SPR technique

Dear all,
Sorry for off-topic question.
I want to study protein interaction with few small molecules using SPR
(Machine- *BIACORE 3000*).

The recombinant protein expressed in bacterial expression system is of 92
kDa (with His tag), pI= 9.

*Question-*
1. What should be the chip of choice- *NTA chip or CM5 chip* of GE
healthcare?
2. Is there any alternative available for chips and machine?

Thanks in advance.

Regards

Praveen

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

[Debasish Kumar Ghosh]

Hi Alex,

In addition to Mirella's suggestion I would like to make an addition which 
might be specifically useful for you. Since your peptide has biotin tag, You 
may use HABA dye assay for the exact quatifiation of biotin (and thus 
biotinylated peptide). As far I recall, Thermo scientific provide a kit for 
this assay. The assay is simple and gives accurate results. 

Best !!!



Debasish

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Alex Lee 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, 06 Feb 2017 03:02:07 +0530 (IST)
Subject: [ccp4bb] How to determine the concentration of biotinylated peptide?

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] For stabilizing protein-protein complex

[Debasish Kumar Ghosh]

Dear Sanjeev,

You can try to clone both of your gene in pET duet vector to coexpress both 
proteins in bacterial expression system and purify with Nickel affinity 
exchange chromatography (both proteins will be his tagged). This will eliminate 
the problem of critical mixing of both proteins in stochiometric ratio as in 
vivo condition (i.e. bacterial cell) will take care of that. 
Other wise, you may determine the stochiometric binding ratio from SPR data 
(like by using Langmuir or other models) and mix the individual proteins 
accordingly. 
Buffer (and ions) play some crucial role in some protein protein interaction. 
So you may also try to play around with different buffer and salt compositions 
(if you intuitively guess the ion or buffer conditions).
As the Kd value is in micro molar range, it seems it is not of very strong 
affinity kind of interaction. So longer incubation (4 degree/ rotation) might 
give at least formation of some complex. 
Hope this helps. 

Best!!



Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: sanjeev kumar <sanjeev@gmail.com>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tue, 31 Jan 2017 21:35:32 +0530 (IST)
Subject: [ccp4bb] For stabilizing protein-protein complex

Dear all,

I am trying to stabilize a protein-protein complex. Our SPR study indicates
it is having micro molar dissociation constant. I tried to purify both the
molecule in complex form with size exclusion chromatography (mixed both the
protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt
observed formation of complex as both the molecule eluted at their
respected elution volume.
Please suggest me to get a better way to achieve the complex and if anyone
gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.

Thanks

best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana

Re: [ccp4bb] Offtopic: Software to closely visualize interacting partnets in protein complex

[Debasish Kumar Ghosh]

Thank you all for your valuable suggestions. They have really worked well for 
me :)

Regards,

Debasish Kumar Ghosh

CSIR- Junior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html

[ccp4bb] Offtopic: Software to closely visualize interacting partnets in protein complex

[Debasish Kumar Ghosh]

Dear All, 

Apologies for this little off-topic inquiry. I want to closely visualize the 
interacting residues in an multimeric protein complex to understand the nature 
of interactions. Is there any good software to give this information with good 
clarity.
Any suggestion is highly appreciated.

Thanks, 
Best !!!

Debasish Kumar Ghosh

CSIR- Junior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html

[ccp4bb] Crystallization of aggregation prone protein

[Debasish Kumar Ghosh]

Dear All,

I am working with a protein which is having an N-terminal unstructured region 
(approximate of length 40-60 amino acids) and C-terminal region which is having 
structured (possibly alpha helical region as probed by CD spectroscopy, 
Bioinformatic studies) property. The protein, in higher concentration (above 
2mg/ml), forms various oligomeric structure (probed by Atomic force microscopy) 
resembling annular type aggregation (may be called as protofibril). when using 
various deletion mutants lacking different regions of N-terminus and 
C-terminus, we found that the C-terminal 45 residues is responsible and 
sufficient to make this annular type aggregation (like seen in alpha Synuclein, 
though much bigger is size than alpha Synuclein annular oligomers). We tried 
with our best to crystallize both the full length protein and the C-terminal 45 
residues (which eventually has 37% sequence identity and 63% similarity [at 
Query coverage of 87%] with a putative UBA domain like structure of an archaeal 
protein). We tried various crystallization suites like PEG, PEG-II, Nucleix, pH 
clear etc to optimize the condition with no satisfaction at all.
So, it would be a great help for me if I can get advises so as to troubleshot 
this problem. All advises are deeply appreciated. 

Thank you,
Best regards,

Debasish Kumar Ghosh

CSIR- Junior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html

[ccp4bb] Crystallization condition for trimeric protein

[Debasish Kumar Ghosh]

Hi,

I am working with a protein which can assume different oligomerization forms, 
starting from monomers to trimers and even penta-decamers. We conformed this by 
Native PAGE and HPLC studies. The protein's theoretical monomeric molecular 
weight is 14.6 KDa (pI - 5.9) and it has some 140 amino acids with high 
Glutamic acid (24), Lysine (10) and Arginine (13) content. I have tried to 
crystallize it but not getting any hit as far now.
Previous study showed that this protein gets some stability by Calcium ion. 
With the calcium chloride conditions, I am getting spherical shaped structures, 
but not sure what are they; calcium chloride crystals or protein crystals. Can 
protein crystals be spherical in shape, specially when the protein behaves like 
an oligomer?
Also please let me know what is the minimum protein concentration required to 
obtain crystal for such small protein (if there is any empirical rule/idea).
Any suggestion will be highly appreciated.

Thanks and regards,
Debasish Kumar Ghosh

CSIR- Junior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088787619 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html