[ccp4bb] Issues with Phenix refinement

[Mohammad Khan]

Dear all,



I am trying to fit Cd in a protein structure using Phenix. However, the
refinement returns me a result with DC (2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE)
in the validation report and gives me the missing atoms accordingly. When I
open the same structure in Coot, it shows me the ion Cd. Also, the .pdb
file contains Cd.



Cd is present in my crystallization condition and fits well into the
density.



Can someone suggest what may be wrong?

Thanks in advance!

Re: [ccp4bb] Off topic: denaturing urea gels

[Mohammad Khan]

Dear all,

Thanks for all the replies. I adjusted my volumes and stopped reactions
with 8 M urea and 25 mM EDTA. The gels now run fine :)

-Mohammad

On 05-Oct-2017 7:29 PM, "Phoebe A. Rice"  wrote:

> Even though the protein should be denatured by all that urea, we find such
> gels sometimes look nicer if stop the reaction with SDS and protease K,
> and/or phenol extract the remains of the protein before loading the
> high-urea gel.
>
> ++
>
> Phoebe A. Rice
> Dept. of Biochemistry & Molecular Biology
> The University of Chicago
> pr...@uchicago.edu
>
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher
> Gileadi [opher.gile...@sgc.ox.ac.uk]
> Sent: Saturday, September 30, 2017 3:44 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Off topic: denaturing urea gels
>
> In addition to the previous suggestions:
>
> With very small gels, the sample composition and depth (in the well) have
> a strong effect on the resolution.
> Rinse the wells with TBE buffer just before loading, as urea from the gel
> diffuses into the well and may prevent the sample from settling at the
> bottom. Minimize the amount of salt in the samples; try to load very small
> volumes (1-3 ul), even if this means longer exposures later.

[ccp4bb] Off topic: denaturing urea gels

[Mohammad Khan]

Dear all,

I am working with an exonuclease and I run the digested DNA on a
8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad
system and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness.
I use a 15 well comb. I run my gels at 70 V for as long as 4 hours till my
undigested DNA reaches half the gel distance. I use 20-30 nt long
susbtrates.

I am mostly not able to get distinct bands of the digested products but
rather get a smear. Is there any way to make sure that I get distinct
digested products rather than a smear?

I am looking forward for suggestions from all!

Thank you.

Ciao!

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Thomas,

I do all my experiments in a 384 well, non-binding plates. I have a salt
concentration of 50 mM NaCl, with BSA. i will try the other suggestions as
well.

>From a couple of experiments, I could determine the Kd to be approx. 10 nM.

Thanks for all the suggestions!



On Fri, Jul 21, 2017 at 4:11 PM, Thomas Edwards <t.a.edwa...@leeds.ac.uk>
wrote:

> A few tips, some/all of which may be relevant. Or not…
>
>
>
> If you are in 96 or 384 well plates, they vary. We tried quite a few
> batches and brands before settling on one.
>
>
>
> Keep your salt concentration as low as possible.
>
>
>
> You should be able to measure Kd in a range of about 1nM to low uM.
> Outside that range is harder, or possibly impossible.
>
>
>
> Buffers matter.
>
> RNA binding proteins have preferred phosphates, PPIs Tris.
>
> Some triton and/or some BSA can help.
>
>
>
> All proteins are different, and each likes some or all or none of the
> above…..
>
>
>
> Ideally you should convert polarization to anisotropy. Simple enough – but
> some referees can get picky…
>
>
>
> *Ed*
>
>
>
> *T.A.Edwards Ph.D.*
>
> *Deputy Director* Astbury Centre for Structural Molecular Biology
>
> Ass. Professor, School of Molecular and Cellular Biology
>
> Garstang 8.53d
>
> University of Leeds, Leeds, LS2 9JT  Telephone: 0113 343 3031
>
> http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
>
> Invention, my dear friends, is 93% perspiration, 6% electricity, 4%
> evaporation, and 2% butterscotch ripple. ~Willy Wonka
>
>
>
> *From: *CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Mohammad
> Khan <mohdkhan0...@gmail.com>
> *Reply-To: *Mohammad Khan <mohdkhan0...@gmail.com>
> *Date: *Friday, 21 July 2017 at 14:32
> *To: *"CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
> *Subject: *[ccp4bb] Off topic: Flourescence anisotropy measurement
>
>
>
> Dear all,
>
>
>
> I am trying to measure the difference in polarization upon the binding of
> the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
>
>
>
> Similar observations have been observed by my colleagues with their
> proteins.
>
>
>
> Are there any tips or precautions to keep in mind while setting up these
> reactions?
>
>
>
> Looking forward for suggestions.
>
>
>
> Thank you.
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Didier and Opher,

The difference in polarities can reach upto 60 units. I have tried
concentrations between 1-5 nM DNA. Maybe I will give higher concentrations
a shot.

Thanks!

On Fri, Jul 21, 2017 at 4:02 PM, Didier Spittler <spittlerdid...@gmail.com>
wrote:

> Hello,
>
> What is your difference between the maximum and minimum value ? Have you
> try to change the probe concentration?
>
> Best,
>
> Didier
>
> Le 21 juil. 2017 15:34, "Mohammad Khan" <mohdkhan0...@gmail.com> a écrit :
>
> Dear all,
>
> I am trying to measure the difference in polarization upon the binding of
> the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
>
> Similar observations have been observed by my colleagues with their
> proteins.
>
> Are there any tips or precautions to keep in mind while setting up these
> reactions?
>
> Looking forward for suggestions.
>
> Thank you.
>
>
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Julius,

That is a good suggestion. I will definitely try this.

Thanks!


On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius <julius.r...@fmi.ch> wrote:

> Dear Mohammad,
> your buffer is key to success in biophysical measurements. While your
> protein may be totally fine in standard buffer, the large and hydrophobic
> fluorophores used in FP, MST and other assays are prone to adsorbing to
> plasticware etc. Also, at 1nM concentration, you are losing much more of
> your protein to adsorption, so you will have far less active protein at
> those concentrations than calculated. I would try to add detergent and, if
> necessary, BSA to your buffer. For my assay development projects, Tween20
> or Brij35 (0.03%) have worked well and I have used 0.5% BSA. Once you get
> the buffer right, measurements will be far more reproducible. Also, try to
> include a good control, e.g. DNA with Cy3, but sequence that does not bind
> to test for nonspecific, concentration dependent effects.
> I hope this helps, if you have any questions, feel free to email me!
> Best,
> Julius
>
>
> > On 21 Jul 2017, at 15:32, Mohammad Khan <mohdkhan0...@gmail.com> wrote:
> >
> > Dear all,
> >
> > I am trying to measure the difference in polarization upon the binding
> of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
> >
> > Similar observations have been observed by my colleagues with their
> proteins.
> >
> > Are there any tips or precautions to keep in mind while setting up these
> reactions?
> >
> > Looking forward for suggestions.
> >
> > Thank you.
>
>

[ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear all,

I am trying to measure the difference in polarization upon the binding of
the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
difference of polarization with decrease in protein concentration. However,
the results are difficult to reproduce and also vary greatly within
triplicates of an experiment.

Similar observations have been observed by my colleagues with their
proteins.

Are there any tips or precautions to keep in mind while setting up these
reactions?

Looking forward for suggestions.

Thank you.

Re: [ccp4bb] Problems with an exonuclease

[Mohammad Khan]

Dear all,

I apologize that till now I was making the mistake of not sending the
replies in the common thread, but somehow only to each person individually.
I am summarizing my answers since yesterday here:

I have tried refolding it at pH 7, 7.5 and 8. I add 4mM TCEP to my urea
buffers and reduce it to 2 mM on refolding. I have tried in presence of DTT
and also in absence of any reductant.
My refolded wild type enzyme shows activity, whereas the mutant doesn't.

Yes, I have got my protein mass-speced. Didn't see any modification!

I have tried to remove the contamination by affinity and ion exchange but
with no avail. I have to try heparin-sepharose though.

I sonicate my samples in the washing steps.
I also suspected Triton X as the contaminant. But I then figured out that
TX-100 has a high absorbance at 280 nm, rather than at 260. I will surely
try the KCl and EDTA suggestions.

I have also tried purifying in absence of TX-100, but with same results!

As for running an agarose gel, I have done so but couldn't really see
anything on an EtBr gel.

I will try the suggestions of washing with other solutions as suggested.

Thank you all for the great suggestions!

On Wed, Jun 7, 2017 at 3:36 PM, Mohammad Khan <mohdkhan0...@gmail.com>
wrote:

> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with
> urea, I refold the protein by rapid dilution and get an aggregate and
> monomer peak of the same on GFC. and have checked CD as well as activity,
> both of which are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>
>

[ccp4bb] Problems with an exonuclease

[Mohammad Khan]

Dear all,

I am working with an exonuclease by refolding it from inclusion bodies
(IBs). I tried various constructs and hosts, but couldn't get it in soluble
form.

I lyse my cells using a cell disruptor and after solubilizing IBs with
urea, I refold the protein by rapid dilution and get an aggregate and
monomer peak of the same on GFC. and have checked CD as well as activity,
both of which are good.

My issues is as follows:

I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
reach upto 2. I have tried all means to get rid of watever this
contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
Dnase prior to lysis. I have also used methods to remove the DNA from
protein, if that is the contaminating agent.
I am trying to crystallize the protein with no success so far.
Moreover, my thermofluor assays give very low fluorescence. I use Sypro
Orange as a fluorophore.

Suprisingly, a point mutation in the active site (His to Arg) gets rid of
the issue of contamination and gives me good thermofluor curves. I purify
the mutant also form IBs.

Can someone suggest what this "contamination" may be?

Thank you for your time.

[ccp4bb] Auto-proteolysis

[Mohammad Khan]

Dear all,

I am working on a His-tag recombinant protein with two domains, which I
purify using affinity chromatography. When I set up crystallization of the
same, it gave me crystals in two different conditions- one was the complete
protein. The other just had the Domain2.

Even on SDS-PAGE, I see three different bands of the purified protein.

How can I determine the autu-proteolysis of this protein? This class of
protein has not reported to have any auto-proteolytic properties, though it
forms isopeptide bonds by autocatalysis.

Thanks a lot

Mohammad

[ccp4bb] Online server for generation of topology cartoons

[Mohammad Khan]

Dear all,

Is there any good online server for the generation of topology cartoons of
proteins, where one can have a clear layout of the various secondary
structures?

I have tried Pro-Origami, but however I am not veru happy with the output.
I have used the default options. Maybe if someone can help me out with
various options.

I would want to use the output for publication purposes.

Thanks

Mohammad

Re: [ccp4bb] Online server for generation of topology cartoons

[Mohammad Khan]

Thanks for the suggestions!

On Wed, May 20, 2015 at 6:21 PM, Mark J van Raaij mjvanra...@cnb.csic.es
wrote:

 There are different servers and programs which I use as guides (TOPDRAW
 comes to mind), but for publication purposes I always end up drawing
 topology diagrams myself - for what I consider maximum clarity. It takes
 time, but I prefer to show things the way I think is best showing them,
 using the output from one or more of these programs as guide. It is than
 also easier to change them upon suggestions of collaborators or referees.

 Mark J van Raaij
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij








 On 20 May 2015, at 11:50, Mohammad Khan wrote:

  Dear all,
 
  Is there any good online server for the generation of topology cartoons
 of proteins, where one can have a clear layout of the various secondary
 structures?
 
  I have tried Pro-Origami, but however I am not veru happy with the
 output. I have used the default options. Maybe if someone can help me out
 with various options.
 
  I would want to use the output for publication purposes.
 
  Thanks
 
  Mohammad
 
 

[ccp4bb] Deletion of hydrogens

[Mohammad Khan]

Dear all,

I have added hydrogens on my molecule using the Refmac option in Coot.
I now want to remove them. I tried various syntax but they didn't
work. Is there a simple way to remove them from the pdb?

Moreover, if I do my refinemnets with hydrogens in it, will it affect
my results, other than maybe slowing down the process.

Thank you

Mohammad