Re: [ccp4bb] Analysis of NMR ensembles

[S. Mohanty]

Hi Harry,
The superpose/overlay of all the structures in PyMol should inform you the 
rigid part of the protein as well as the flexible part. The rigid part would 
have very low backbone RMSD or overlay tightly and the flexible part (loops, 
N-term and C-term etc.) would not superpose tightly. If you check literature, 
the dynamics of the protein may have been studied through NMR relaxation. 
Smita 

On Wednesday, May 26, 2021, 10:05:05 AM CDT, Harry Powell - CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:  
 
 Hi

Given that there are plenty of people on this BB who are structural biologists 
rather than “just” crystallographers, I thought someone here might be able to 
help.

If I have a structure in the PDB (e.g. 2kv5) that is an ensemble of structures 
that fit the NOEs, is there a tool available that will give me some idea about 
the bits of the structure that do not vary much (“rigid”) and the bits that are 
all over the place (“flexible”)?

Would superpose or gesamt be a good tool for this? Ideally I’d like something 
that could add a figure to the B columns in a PDB file so I could see something 
in QTMG (or PyMol if forced…) or do other useful things with the information.

Harry


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Re: [ccp4bb] protein oligomer

[S. Mohanty]

Keep the protein concentration low during purification steps along with using 
other anti-aggregation agent/s. Make sure that the pH at which you are 
purifying is not close to the pI of the protein. Until completely purified, all 
purification steps should be performed in a cold room if it is a soluble 
protein.
Smita  

On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga 
 wrote:  
 
 I am not sure what you mean by polymer formation. Presuming that you have 
optimized your protein concentration, pH and salt concentration, you could try 
arginine as an anti-aggregation agent in your purification (I presume you do 
FPLC). Have a look at chaotropic agents used in protein purification, The 
answer is generally dependent on the protein/proteins you are trying to purify 
and is not necessarily straightforward.
Kinds regards
On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn> wrote:


 Dear all:

 Any ideas to decrease protein polymer formation? my protein was easy to form 
oligomers and precipitation when do purification,I have tried add glycerol and 
DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks!

 Best Regards


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Re: [ccp4bb] Difficult purification with imac columns

[S. Mohanty]

Hi Narayanan,We had similar problems with a membrane protein. It did bind 
Ni-NTA resin strongly. We washed out the resin (Ni-NTA) with high imidazole to 
sealer all other proteins and eluted out protein with very small concentration 
of SDS at room temp with shaking. Please read our paper in- Huang et al., 
Biochemistry, 49, 1115-1126, 2010Hope this procedure can help you too.Smita  

On Friday, September 15, 2017 6:54 AM, Narayanan Ramasubbu 
 wrote:
 

 Hi. We are working on a periplasmic protein that breaks naked glycans in 
peptidoglycans. There is truncated structure available but our target is the 
full length protein. The difficulty us that it strongly binds to the resin with 
or without his.tag. Changing the resin to acrylamide did not help. 
Has anyone come across similar problem and how was it resolved. 
The pdb structure is the catalytic domain and mussing a region that, in my 
opinion, binds to the resin.  
Thank you in advance
Sent from my iPhone

   

Re: [ccp4bb] Problems with an exonuclease

[S. Mohanty]

Yes, washing IB with 10 % BPER  twice with sonication makes our IB (from 
various proteins) very clean before denaturation with 6M guanidine 
hydrochloride and further processing. Smita  

On Wednesday, June 7, 2017 12:03 PM, Nicole Thomas  
wrote:
 

 I've found that washing my IB's with B-PER helps dramatically to get rid of 
any impurities.
https://www.thermofisher.com/order/catalog/product/78248

Nicole ThomasUniversity of Wisconsin, MadisonGellman Group
On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers  
wrote:

I missed the Triton - that will be it!
On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers; 1. 20mM Tris, 500mM NaCl, 1% 
Triton-X-100, pH 7.52. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.53. 10mM 
Tris, 1M NaCl4. 20mM Tris, 500mM NaCl, pH 7.5 By resuspension and then 
sonication. This I find removes DNA and Triton-X-100. Also, if the pellet is 
very large, you may need to increase the number of washes, volume and length of 
sonication or split the pellet up. Other things to try…1.  Change the wash 
salt to KCl and use more, (3M). I was informed that KCl is a better disrupter 
of DNA than NaCl (I stand to be corrected if this is wrong).2.  At each 
wash stage, dissolve a small amount of IBs and measure the 260/280. The ratio 
should decrease in the latter washes, if they are working.3.  Does your 
exonuclease typically contain a divalent metal? You could try adding EDTA to 
the wash steps which may help in preventing DNA stick to your protein. All the 
best! Dan  Daniel A Bonsor PhD.Sundberg LabInstitute of Human 
VirologyUniversity of Maryland, Baltimore725 W Lombard Street 
N370BaltimoreMarylandMD 21201Tel: (410) 706-7457  From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK]On Behalf Of Mohammad Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease 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.  



-- 
Best wishes
Prof. Jon R Sayers, FRSBTel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.ukhttp://www.sheffield.ac.uk/ iicd/profiles/sayers





   

Re: [ccp4bb] Protein precipitation

[S. Mohanty]

Hi Manjula,
I will lyse the bacterials cells and monitor the degradation of the lysed 
protein in lysis buffer over 4-5 days before proceeding with purification.  
What is the lifetime of this expressed protein once lysed?  How long can it 
stay after lysis without degradation or aggregation? 
 Does this protein contain disulfide bonds?  How many?  How big is this protein?
Smita Smita Mohanty, Ph.D.  Associate Professor Department of Chemistry447 
Physical SciencesOklahoma State UniversityStillwater, OK 74078-3071 Phone: 
(405) 744 6636E-mail: Smita.Mohanty@okstate.eduWeb: 
chemistry.okstate.edu/mohanty                            


 On Monday, May 18, 2015 11:49 PM, Manjula Ramu manjula@gmail.com 
wrote:
   

 Hi all,I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 
mm NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed 
affinity purification. Eluted with 200mM imidazole.  While elution I could see 
slight turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 
6.0, here also same problem I faced.Please suggest me a method where I can get 
stable protein.
Thanks and Regards,Manjula R 
Research Scholar
Department of Biophysics 
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula.iob@gmail.comMobile no:+91-9538553356
http://www.nimhans.kar.nic.in/


  

[ccp4bb] Postdoc position in Structural Characterization of a membrane-associated enzyme

[S. Mohanty]

Postdoc position in Structural Characterization of a membrane-associated enzyme
Lookingfor an outstanding and highly motivatedindividual to start immediately 
in a postdoctoral position to study structureand mechanism of a 
membrane-associated enzyme involved in co-translationalmodification. The 
project aims to elucidate the mechanistic and structuralbasis for the enzyme 
function using a multi-disciplinary approach comprisingX-ray crystallography, 
NMR spectroscopy, and biochemical methods.The candidate will focuson cloning, 
overexpression, purification, biochemical characterization,crystallization, and 
X-ray crystal structure determination of multiple proteinsubunits and 
protein-substrate complexes.

The candidate should havea recent Ph.D. degree (in biochemistry, biophysics, 
structural biology orrelated area) with a strong publication record. In 
addition, the candidate isrequired to have substantial experience in protein 
expression specifically inyeast, membrane protein purification and 
reconstitution and proteincrystallography. Proficiency is required in 
troubleshooting and optimization ofcloning, overexpression and reconstitution 
of membrane proteins protocols. Ahigh level of motivation and good 
communication skills are required. Competitivesalary and fringe benefit will be 
provided. 
Requests for more information aswell as application that should 
include a cover letter stating your scientificinterests and goals, CV and 
contact information for 3 references can be sent toAssociate Professor Smita 
Mohanty at smita.moha...@okstate.edu.     Institution: Oklahoma StateUniversity 
  Location:   Stillwater,OK StartDate:  Immediately Contact:Smita Mohanty, 
Ph.D.                    Associate Professor
                    Department of Chemistry
                    Oklahoma State University, Stillwater, OK                   
  E.mail:smita.moha...@okstate.edu