[ccp4bb] E.coli RNA polymerase

[vipul panchal]

Dear All,

First of all, excuse me for writing an off-topic subject.

Our group is working on high throughput screening of a non-coding RNA to
investigate its therapeutic potential. We require *E.coli* RNA polymerase
and *E.coli* sigma-70 to perform In-vitro transcription for bulk synthesis
of ncRNA.
NEB has offered us a price of 84,000 EUR for 50ml of E.coli RNA pol
saturated with sigma-70. This is still expensive for us.

I am writing here hoping for a potential collaboration with a group who
routinely purifies *E.coli* RNA pol and sigma-70. If this is possible,
kindly let me know.

Best wishes,
Vipul
-- 
Vipul Panchal, PhD
University of Bergen
(M)-+47 46359545



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

[vipul panchal]

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, <
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*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] translational NCS & twinning

[vipul panchal]

Hi Donghyuk,
I feel you went ahead with right strategy.
For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong
indicator, i believe.
If you have already build all possible model, using tls can be of further
help.

Cheers,
Vipul




On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin  Dear all,
>
> I am having tough time with my Xtal data sets those seem to be twinned or
> have translational NCS, and it will be greatly appreciated if you can give
> me some advices or comments!
>
> Data was initially processed with XDS and scaled with aimless without
> specifying certain space group (SG).
> Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates
> there is non-origin peak after patterson analysis. (attached log)
> And, I could not get the proper MR-solution from this data sets.
>
> Because I read that twinning and tNCS cannot be properly distinguished at
> high SG, I went down to subgroup either P32 or P6 assuming that there is
> twinning which make data set seems to have apparently high SG. (procedure
> was same XDS->aimless but I specified the SG to keep them)
> Then, xtriage still indicates there is non-origin peak as before, but
> found twin laws for the data sets (attached log).
> However, I still could not get the right MR-solution.
> Then, I went even further down to P3 or C2, and xtriage found more twin
> laws which is make sense because of the lower SG. (attached log)
> Again, I could not get the MR-solution.
> For all the MR running above, I assumed that phaser(ccp4 module)
> automatically applied tNCS if they present. or should I have to tick on
> button in the expert parameters?
>
> Then, I went back to the image and processed the datasets with mosflm by
> checking the indexed spots.
> During this step, I played with the threshold for indexing to follow the
> strong spot for get correct SG.
> I am not sure whether this is correct or not, but by putting high
> threshold for indexing (e.g. ~15) I could index the data with C2 which has
> half dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the
> original unit cell (232.533, 134.202, 182.67, 90, 90, 90).
> With this, I could put 3 molecules in ASU by MR. During refinement, I felt
> that the R values were not dropping, and I applied twin refinement.
> without twin refinement the R values were (0.39/0.44, work/free), and
> applying twin refinement gave me significantly better values (0.23/0.26).
> Because there were 6 twin operators which may cause this huge R value
> drop, I speculate whether this is true or not.
>
> Your comments will be greatly helpful!
>
> With you all the best,
> Donghyuk
>
>
>
> 
>
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Re: [ccp4bb] cavities in protein structures

[Vipul Panchal]

Hi Claudia,
For the identification of cavities and residues linning them, metapocket is
one of the preferred choice as it uses prediction from various program.
Output of the server is pdb file.

I found caver program represent cavities in a best manner. There is caver
plugin available for pymol. Output is tunnel like representation with
different colours. Interestingly, it also provides tunnel length, radius
and list of residues linning the bottle-neck of each of the cavity. As a
input for the presentation of cavities, you need to provide list of
residues lining cavities. Identified by other programs.

All the best!


On 30-Jan-2018 6:28 PM, "Boaz Shaanan"  wrote:

Hi Claudia,

Another possibility is CastP: http://sts.bioe.uic.edu/castp/index.html

They also have a Pymol plugin. I have not used this plugin since I'm
displaying the CastP o/p files in UCSF-chimera which handles them nicely.


Cheers,


Boaz












*Boaz Shaanan, Ph.D. Dept. of Life
Sciences  Ben-Gurion University of the
Negev  Beer-Sheva
84105
Israel
E-mail:
bshaa...@bgu.ac.il  Phone: 972-8-647-2220  Skype:
boaz.shaanan  Fax:   972-8-647-2992 or 972-8-646-1710*







--
*From:* CCP4 bulletin board  on behalf of Claudia
Binda 
*Sent:* Tuesday, January 30, 2018 1:51 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] cavities in protein structures

Hi everyone,

I need suggestions to calculate and represent cavities of protein
structures. For years I have been using Voidoo that produces maps in ezd
format which could be converted in map format (ccp4) using the online
server http://xray.bmc.uu.se/cgi-bin/gerard/mapman_server.pl. However, this
does not work anymore. Is there another way to do it? What is the best tool
to calculate cavities and draw them by Pymol or ccp4mg?

Thank you
Claudia







-- 
Claudia Binda
University of Pavia
Dept. Biology and Biotechnology
via Ferrata 1, 27100 Pavia - Italy
Phone: +39-0382-985535 <+39%200382%20985535>
Fax: +39-0382-528496 <+39%200382%20528496>
E-mail: claudia.bi...@unipv.it
Web: http://www.unipv.it/biocr 

Re: [ccp4bb] RMSD between superposed structures without moving

[Vipul Panchal]

May be you should try pdbefold server. It provides rmsd between equivalent
CA atoms of superimposed structures.

All the best.
Vipul Panchal,
Ph.D. student
CSIR-IGIB
(M)- 091 7678617949

On 27-Aug-2017 4:50 PM, "Johannes Sommerkamp" <
155b9e78396e-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello everybody,
> I have superposed two structures based on the central beta-sheet CA atoms
> with the "super" command in Pymol.
> Now, I want to calculate the RMSD between ALL atoms or ALL CA atoms
> without moving the structures again. The rms_cur command in Pymol would do
> that, but only works if all atom identifiers match. Adding "transform=0" to
> the super, oder align command still does the alignment and moves the
> structure but does not show the movement.
>
> Is there an easy way to just calculate the all atom RMSD between two
> already superposed structures in pymol or any other programm?
>
> Thanks in advance!
> Johannes
>
> --
> Johannes Sommerkamp
> Ruhr-Universität Bochum
> AG Röntgenstrukturanalyse an Proteinen, LS Biophysik, ND04/396
> Universitätsstraße 150
> 44801 Bochum
> Tel: +49-(0)234/32-25754
>

Re: [ccp4bb] Ramachandran oultliers increase upon restrained refinement

[Vipul Panchal]

I do agree with Eleanor. When I was solving structure at 2.16A resolution,
outlier residues were having stearic clash or required side chain flipping.

At 2.76A resolution, hydrogen bonds would be difficult to visulize.

I found phenix.refine more user friendly. You may too find it useful.

Vipul Panchal,
Ph.D. student
CSIR-IGIB
(M)- 091 7678617949

On 02-Jul-2017 9:14 PM, "Eleanor Dodson"  wrote:

Just remember - most Ramachandran outliers are due to errors in the
environment - eg: maybe some side chains clash hence refinement tries to
move things to accommodate that bad feature, etc etc etc...
At that resolution it is inevitable you will have some level of
mis-interpretation ..
4% outliers does not surprise me.

EDSTATS is a useful tool to find things such as pep flips - you can access
it most simply from CCP4 GUI2 .

But unless the outliers are in a important part of the structure I would
suggest checking, then living with them.

Eleanor






On 2 July 2017 at 16:31, Rajesh Kumar  wrote:

> Dear Meytal,
>
> PHENIX-REFINE has an option for Ramachandran outlier refine. If you check
> this on, it will do the work. But after this refinement, you must check
> every residues to make sure residues are in the density.
>
> Thank you
> Rajesh
>
>
>  ---x
> With regards
> Rajesh K. Harijan, Ph.D.
> Schramm Laboratory
> Albert Einstein College of Medicine
> 1300 Morris Park Ave., Bronx, NY 10461
> Tel: 718.430.2777 <(718)%20430-2777>  |  Fax: 718.430.8565
> <(718)%20430-8565>
>
>
>
> On Sun, Jul 2, 2017 at 7:26 AM, Meytal Galilee 
> wrote:
>
>> Dear all,
>> I am refining a 2.76A structure using refmac5.
>> I keep fixing the Ramachandran outliers down to 18 (1.9%), however, upon
>> restrained refinement, the outliers increase back to 35.
>> Do you have any suggestions how can I fix my structure?
>> Thanks,
>> Meytal Galilee
>>
>>
>
>
>

Re: [ccp4bb] Bond length and angle outlier fixing

[Vipul Panchal]

Thanks all for the reply. I will follow your suggestion and will get back.

Sincerely,

On Thu, May 18, 2017 at 5:02 AM, Paul Adams  wrote:

> To add to Pavel’s comments, it is also possible to do the automated model
> building in Phenix:
>
> http://www.phenix-online.org/documentation/reference/map_
> to_model.html
>
> As Pavel said: there is Phenix mailing list for Phenix-specific questions
>
>
> > On May 17, 2017, at 10:24 AM, Paul Emsley 
> wrote:
> >
> > Hi, all.
> >
> > I have collected cryoEM data and want to use Coot and CCP4 program to
> build model and to refine it.
> >
> > 1. What is the steps to do this?
> > 2. How do I convert cryoEM map file to MTZ file?
> > 3. Can I also use Phenix for this purpose?
> >
> > Thanks to all for your help in advance.
>
> --
> Paul Adams
> Division Director, Molecular Biophysics & Integrated Bioimaging, Lawrence
> Berkeley Lab
> Division Deputy for Biosciences, Advanced Light Source, Lawrence Berkeley
> Lab
> Adjunct Professor, Department of Bioengineering, U.C. Berkeley
> Vice President for Technology, the Joint BioEnergy Institute
> Laboratory Research Manager, ENIGMA Science Focus Area
>
> Building 33, Room 347
> Building 80, Room 247
> Building 978, Room 4126
> Tel: 1-510-486-4225, Fax: 1-510-486-5909
> http://cci.lbl.gov/paul
>
> Lawrence Berkeley Laboratory
> 1 Cyclotron Road
> BLDG 33R0345
> Berkeley, CA 94720, USA.
>
> Executive Assistant: Louise Benvenue [ lbenve...@lbl.gov ][
> 1-510-495-2506 ]
> --
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] Bond length and angle outlier fixing

[Vipul Panchal]

HI all.

I am solving protein structure with 2.16A resolution. There are two chain
in an asymmetric unit.
While submitted to PDB validation server, i could see few ligand bond
-length and -angle outlier. Coot doesn't have any module that can help me
with these as per my best understanding.
Kindly find the image of relevant details attached herewith.

Can somebody suggest me how to fix them?

Sincerely,
-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Poor density fit.

[Vipul Panchal]

Thanks Bert,

I did understand not to delete such atoms or set occupancy to zero from
forum and literature. However, colleagues in the vicinity informed to take
approaches i mentioned earlier. Therefore, I thought to take opinion from
forum if something has changed recently.

Thanking,



On Thu, May 4, 2017 at 8:55 PM, Bert Van-Den-Berg <
bert.van-den-b...@newcastle.ac.uk> wrote:

> This has been discussed before, I guess more than once
>
> I think most people (I'm sure i'll be corrected if wrong) would favor not
> removing any atoms or setting occupancies to zero and let the invisible
> atoms be accounted for by high B-factors (either set manually or just
> letting refinement do its thing).
>
>
> Bert
>
>
> ------
> *From:* CCP4 bulletin board  on behalf of Vipul
> Panchal 
> *Sent:* 04 May 2017 16:12
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Poor density fit.
>
> HI all.
>
> I am solving protein structure with 2.16A resolution. There are two chain
> in an asymmetric unit. I see that in one of the chain, many residues'
> density for side chains is incomplete and therefore results in poor density
> fit.
>
> *I want to know your opinions for the approach I have taken. Figures
> relevant to each approach have been attached herewith.*
>
> *Case1*: There is no experimental density at all. Therefore, i have
> deleted side chains to Gly.
> *Case2*: Though there is incomplete density for Leu, it is enough to
> suggest its rotamer. In this case, as may be seen, i have just set
> occupancy for atoms without density(CG, CD1, CD2) to zero.
>
> Hopeful for the response.
>
> --
> Vipul Panchal
> Senior Research Fellow,
> Respiratory disease and biology,
> CSIR-IGIB
> (M)-9540113372
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb] Poor density fit.

[Vipul Panchal]

HI all.

I am solving protein structure with 2.16A resolution. There are two chain
in an asymmetric unit. I see that in one of the chain, many residues'
density for side chains is incomplete and therefore results in poor density
fit.

*I want to know your opinions for the approach I have taken. Figures
relevant to each approach have been attached herewith.*

*Case1*: There is no experimental density at all. Therefore, i have deleted
side chains to Gly.
*Case2*: Though there is incomplete density for Leu, it is enough to
suggest its rotamer. In this case, as may be seen, i have just set
occupancy for atoms without density(CG, CD1, CD2) to zero.

Hopeful for the response.

-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] very high B-factor

[Vipul Panchal]

1) the difference is 5.23 % and it is good and acceptable.
2) if higher value belongs to residue, then you need to change the rotamer
Using coot ,  follow menu-->distance-->residue info. You will get to know
to whom 417 B factor value belongs. Meanwhile, just check if modeled
residue or part of residue has relevant density. If density is missing for
part of residue, then set occupancy to zero for the relevant atom(s). If
density is missing for the model of residue, then just remove side chain. I
would recommend to check density fit (coot-->menu--> validate--> density
fit) for all residues with >50 B factor value (coot-->menu--> validate-->
temp. fact. variance analysis).


On 04-May-2017 3:26 PM, "yanqiaoling2782048" 
wrote:

Hi Vipul,

Thanks for your quick reply.

1. Actually, i mainly comparing the gap between Rwork and Rfree which
should be <5%, and then the overall b-factor, RMSD of bond and angle. For
me, no5 is the best one, but the "B factor variance Graphs" make me
uncomfortable.
The R-factor gap of no6 is 6.23%, does this mean overfitting?

2. The b-factor value of 76 is belong to the atom of the residue obtained
from the pdb file. I'm not sure whether the B-factor in  "B factor variance
Graphs" belong to residue or atom, but i guess 417 belongs to residue.
What's the relationship of the two factor?

Best regards,
Qiaoling Yan


At 2017-05-04 15:58:36, "Vipul Panchal"  wrote:

Hi,

1>
Well, it seems you are just comparing Max value across all protocols. You
should compare average values as it also takes into account no. of atoms
with given values of B factor. As per refinement results, it seems though
no. 6 has highest B factor value, number of atomic outliers are relatively
low. So if you ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you
should go ahead with no.6 is strategy.

2>
When comparing B factor values of 417 vs 76, you should consider what it
belongs to. As i may guess, 417 value belongs to an atom of residue whereas
76 belongs to residue. Meaning 417 is individual B factor whereas 76 is
grouped B factor.


On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048 <
yanqiaoling2782...@126.com> wrote:

> Dear all,
>
> I'm working on a crystal structure with resolution of 2.2A. At the final
> step, I use different strategies to refine the structure, they are:
> no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
> results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)
>
> no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 /
> optimize_xyz/adp_weight=true
> results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/m
> ax/average)
>
> no6: strategy=individual_sites+individual_adp / anisotropic for all
> residues and isotropic for water /
>  set_b_iso=10 / optimize_xyz/adp_weight=true
> results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7
> (min/max/average)
> PS: the results is read from pdb file
>
> The results showed that the strategy of no5 is the best one. But the "B
> factor variance Graphs" generated by coot with
> menu/validate/temp.fact.variance analysis, have shown that no6 have the
> lowest B-factors (the attached figure is the B-factor graphs of three pdb
> files). And my questions are:
> 1. Which strategy should I choose to refine my structure? Or any other
> suggestions to refine the structure at 2.2A resolution?
> 2. Does it possible that some residues have very high B-factor in "B
> factor variance Graphs", while in the pdb file， the b-factor of
> corresponding residues are relatively low? For example, one residue have
> B-factors of 417 in "B factor variance Graphs", but in PDB file the b
> factor is 76. Does the two factor mean the same thing?
> 3. If i want to set the weight manually, which parameter should i set,
> wxc/wxc_scale? or others?
>
> Thanks in advance.
>
> Best regards,
> Qiaoling Yan
>
>
>
>
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] very high B-factor

[Vipul Panchal]

Hi,

1>
Well, it seems you are just comparing Max value across all protocols. You
should compare average values as it also takes into account no. of atoms
with given values of B factor. As per refinement results, it seems though
no. 6 has highest B factor value, number of atomic outliers are relatively
low. So if you ask me no. 6 is the best one.
Coot results are also in congruent with refinement results. I think you
should go ahead with no.6 strategy.

2>
When comparing B factor values of 417 vs 76, you should consider what it
belongs to. As i may guess, 417 value belongs to an atom of residue whereas
76 belongs to residue. Meaning 417 is individual B factor whereas 76 is
grouped B factor.


On Thu, May 4, 2017 at 9:08 AM, yanqiaoling2782048 <
yanqiaoling2782...@126.com> wrote:

> Dear all,
>
> I'm working on a crystal structure with resolution of 2.2A. At the final
> step, I use different strategies to refine the structure, they are:
> no4: strategy=individual_sites+individual_adp+tls / set_b_iso=20
> results: Rwork/free=0.2052/0.2658  b-factor=11.4/136.8/48(min/max/average)
>
> no5: strategy=individual_sites+individual_adp+tls / set_b_iso=10 /
> optimize_xyz/adp_weight=true
> results: Rwork/free=0.2161/0.2639  b-factor=11.3/135.2/48.4(min/
> max/average)
>
> no6: strategy=individual_sites+individual_adp / anisotropic for all
> residues and isotropic for water /
>  set_b_iso=10 / optimize_xyz/adp_weight=true
> results: Rwork/free=0.2183/0.2706  b-factor=10.9/144.6/45.7
> (min/max/average)
> PS: the results is read from pdb file
>
> The results showed that the strategy of no5 is the best one. But the "B
> factor variance Graphs" generated by coot with 
> menu/validate/temp.fact.variance
> analysis, have shown that no6 have the lowest B-factors (the attached
> figure is the B-factor graphs of three pdb files). And my questions are:
> 1. Which strategy should I choose to refine my structure? Or any other
> suggestions to refine the structure at 2.2A resolution?
> 2. Does it possible that some residues have very high B-factor in "B
> factor variance Graphs", while in the pdb file， the b-factor of
> corresponding residues are relatively low? For example, one residue have
> B-factors of 417 in "B factor variance Graphs", but in PDB file the b
> factor is 76. Does the two factor mean the same thing?
> 3. If i want to set the weight manually, which parameter should i set,
> wxc/wxc_scale? or others?
>
> Thanks in advance.
>
> Best regards,
> Qiaoling Yan
>
>
>
>
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

[ccp4bb]

[Vipul Panchal]

Provided your protein is >90 pure as per 15% SDS-PAGE analysis, you should
try seeding.

On 28-Apr-2017 12:43 PM, "李霞"  wrote:

Dear all:

My protein is a demethylase,has failed to obtain protein crystals,then
adopt the method of in situ enzyme and get protein crystals,but the crystal
is still very very small,how to optimize can get diffraction
crystal,please?


Best

X. L

Re: [ccp4bb] AW: [ccp4bb] NCS difference

[Vipul Panchal]

Hi Schreuder,

Thank you for your suggestion.
As per the electron density, the conformation is very obvious. So I think
whatever NCS outliers are there in structure, are real ones.

Thanking you all,

On 24-Apr-2017 1:29 PM,  wrote:

> Dear Vipul,
>
>
>
> At this resolution and with these Rfactors you are not supposed to
> „correct“ the NCS outliers. Look into the electron density maps if they are
> well defined and if the different conformations can be explained by e.g.
> crystal contacts.
>
>
>
> However, if they are in a less well-defined region, you should superimpose
> the NCS symmetry molecule(s) and see which conformation would fit best and
> fit this conformation in the other molecules as well.
>
>
>
> Best,
>
> Herman
>
>
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von
> *Vipul Panchal
> *Gesendet:* Montag, 24. April 2017 09:49
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [ccp4bb] NCS difference
>
>
>
> Hi all,
>
>
>
> I am solving structure of one of the acyltransferse protein. We have
> collected data at 2.16A resolution. Currently the Rfree is 0.2508 and
> Rwork is 0.2042.
>
>
>
> *My query is regarding NCS difference.* Under validation tool of coot
> while looking for NCS differene, i can find some residues with red bar. *Can
> some one suggest me how may i minimize it if i am expected to do it?*
>
>
>
>
> --
>
> Vipul Panchal
>
> Senior Research Fellow,
>
> Respiratory disease and biology,
>
> CSIR-IGIB
>
> (M)-9540113372
>

Re: [ccp4bb] NCS difference

[Vipul Panchal]

I am not from any structure biology related group. So I have followed
literatures​ for the structure solution and analysis. At this level all
stereochemical and geometry looks good. The only parameter I could see with
some red bar was NCS difference.
*Through literature, I couldn't understand if some red bars in NCS
DIFFERENCE window should be a concern.*
So it would be helpful if some of you can suggest me some relevant
literature or practice regarding NCS difference.


On 24-Apr-2017 1:25 PM, "Pavel Afonine"  wrote:

Minimizing a red bar may be tricky.. Have you tried to make it less red
(blue or may be green)? Otherwise Rw/Rf~20/25 is just fine at 2.2A
resolution. What exactly your worry is about?
Pavel

On Mon, Apr 24, 2017 at 12:48 AM, Vipul Panchal 
 wrote:

Hi all,

I am solving structure of one of the acyltransferse protein. We have
collected data at 2.16A resolution. Currently the Rfree is 0.2508 and Rwork
is 0.2042.

*My query is regarding NCS difference.* Under validation tool of coot while
looking for NCS differene, i can find some residues with red bar. *Can some
one suggest me how may i minimize it if i am expected to do it?*


-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372




On 24-Apr-2017 1:25 PM, "Pavel Afonine"  wrote:

> Minimizing a red bar may be tricky.. Have you tried to make it less red
> (blue or may be green)? Otherwise Rw/Rf~20/25 is just fine at 2.2A
> resolution. What exactly your worry is about?
> Pavel
>
> On Mon, Apr 24, 2017 at 12:48 AM, Vipul Panchal 
> wrote:
>
>> Hi all,
>>
>> I am solving structure of one of the acyltransferse protein. We have
>> collected data at 2.16A resolution. Currently the Rfree is 0.2508 and
>> Rwork is 0.2042.
>>
>> *My query is regarding NCS difference.* Under validation tool of coot
>> while looking for NCS differene, i can find some residues with red bar. *Can
>> some one suggest me how may i minimize it if i am expected to do it?*
>>
>>
>> --
>> Vipul Panchal
>> Senior Research Fellow,
>> Respiratory disease and biology,
>> CSIR-IGIB
>> (M)-9540113372
>>
>
>

[ccp4bb] NCS difference

[Vipul Panchal]

Hi all,

I am solving structure of one of the acyltransferse protein. We have
collected data at 2.16A resolution. Currently the Rfree is 0.2508 and Rwork
is 0.2042.

*My query is regarding NCS difference.* Under validation tool of coot while
looking for NCS differene, i can find some residues with red bar. *Can some
one suggest me how may i minimize it if i am expected to do it?*


-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Pymol point mutagenesis turns out to be deletion of a residue

[Vipul Panchal]

I am not experienced with pymol. However if you are familiarized with coot,
you can mutate and set rotamer. It is really simple.

On 12-Apr-2017 12:15 AM, "Alex Lee"  wrote:

> Dear All,
>
> I am using MacPymol 1.8.6, I did Pymol point mutagenesis using Wizard,
> everytime after I choose a residue for mutation, choosing a mutated target
> residue and a rotamer and click apply, the text of the sequence on top of
> the main window shows my selected residue is mutated but the graphics in th
> main window shows deletion of the residue (a broken empty space between
> flanking residues of the mutated residue).
>
> I do not know if any of you experience similar problems.
>
> Thanks for any input.
>

Re: [ccp4bb] Accidental crystallization of E. coli protein

[Vipul Panchal]

I don't think it is going to be any scientific story. Even if you think of
publishing what aspect are you going to discuss? What new are you going to
give to the community.

On Wed, Apr 5, 2017 at 2:46 AM, Mohamed Noor 
wrote:

> During the crystallization of a totally unrelated protein from a different
> bacterium in E. coli, we managed to somehow crystallize an E. coli protein.
> It turned out to be only the catalytic domain of an enzyme. Two previous
> reports both used recombinant expression of this enzyme followed by limited
> proteolysis in order to crystallize this domain.
>
> Has something like this been reported before? I know the stories of AcrB
> etc., but I am looking specifically for a 'naturally proteolyzed'
> crystallization.
>
> Looking at our structure and the previously published one, there is not
> much difference, with an rmsd of about 0.3 A but our data resolution is a
> bit higher. This is perhaps unsurprising as the unit cells are very similar
> (in fact, I just did a quick RBR). Should we bother depositing and
> publishing this observation?
>
> Thanks.
> Mohamed
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[Vipul Panchal]

There is also lack of information here.  Do you really expect this protein
in soluble fraction? Is it a membrane associated or transmembrane protein?

Well, I don't have any experience in protein expression using codon
optimization.
However, Considering the fact that in E.coli it is getting expressed in
insoluble fraction, I would recommend to co-express this protein with
groEL/ES system. I have plasmid expressing groEL/ES operon.

On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant <
g.hans...@dkfz-heidelberg.de> wrote:

> Hi,
>
> Why don't you try adding GST/MBP tags first? This is a easy quick test.
>
> We have a nice fusion (MBP) vector for e.coli expression if you want.
>
> Grant
>
> From: Sutapa Chakrabarti  chakr...@zedat.fu-berlin.de>>
> Reply-To: Sutapa Chakrabarti  chakr...@zedat.fu-berlin.de>>
> Date: Monday 3 April 2017 08:49
> To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" <
> CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>
> Subject: [ccp4bb] Using a codon-optimised gene to improve protein
> solubility
>
> Dear All,
>
> We're trying to express and purify a 1000 residue long protein and have
> run into the problem that it is completely insoluble when expressed in
> E.coli and is not expressed at all in insect cells. The usual tricks for
> improving solubility in E.coli, such as addition of GST/MBP tags,
> optimising expression media and induction conditions and use of different
> cell strains, have not led to any improvement.
>
> We are now looking into ordering a codon-optimised synthetic gene for this
> protein and are trying to decide whether it would be worthwhile to
> codon-optimise for expression in E.coli (given that the protein was
> expressed but not soluble) or if we should attempt baculovirus expression
> again with a gene that has been codon-optimised for insect cells.
>
> My question is:
> has anyone observed an improvement in the solubility of their target
> protein using a codon optimised gene?
>
> I know of several instances where the use of a codon-optimised gene has
> led to expression where the native gene sequence did not but am unable to
> find any references for improvement in solubility. Since codon optimisation
> significantly alters the translation rate of a gene, I believe this should
> affect solubility as well; but I'd like to know what the community
> thinks/has observed before I order an exorbitantly priced gene!
>
> Thank you in advance,
> Sutapa
>
> --
> Sutapa Chakrabarti, Ph.D.
> Institute of Chemistry and Biochemistry
> Freie Universität Berlin
> Takustr. 6
> 14195 Berlin
> Germany
> Phone: +49-(0)30-83875094
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372