Re: [ccp4bb] Need suggestion

Dear Afsan, Based on the information you provided, it's possible that the crashing issue in COOT when attempting to merge the monomers could be due to several reasons. Here are a few suggestions and potential workarounds you can try: Either Update COOT or Import monomers as coordinates: Instead

Re: [ccp4bb] Need suggestion

On 27/06/2023 15:01, Afshan Begum wrote: Dear expert, I am currently working on building my cryo-EM data using COOT. However, I have encountered an issue when trying to add monomers such as Glycerol or BCT using their letter code from the "Get monomer" option in COOT. Whenever I attempt to

[ccp4bb] Need suggestion

Dear expert, I am currently working on building my cryo-EM data using COOT. However, I have encountered an issue when trying to add monomers such as Glycerol or BCT using their letter code from the "Get monomer" option in COOT. Whenever I attempt to merge them into my coordinate, COOT crashes. 

Re: [ccp4bb] Need suggestion for protein solubility

roysi...@gmail.com On Monday, December 26, 2016 2:29 PM, Mark J van Raaij wrote: if you can figure out what is making your protein precipitate, you could put something in the collection tubes of the NiNTA column to prevent precipitation. For example a bit of

Re: [ccp4bb] Need suggestion for protein solubility

if you can figure out what is making your protein precipitate, you could put something in the collection tubes of the NiNTA column to prevent precipitation. For example a bit of concentrated buffer to change the pH, or EDTA. Another thing, you say your protein is pure after NiNTA, but it may

Re: [ccp4bb] Need suggestion for protein solubility

Hi, Regarding the second publication below on the optimum solubility screen, I was a new postdoc in Sung-Hou Kim's lab when the last parts of the work and manuscript was being prepared, and so did quite a few experiments using the screen in conjunction with DLS. It is worth trying them out

Re: [ccp4bb] Need suggestion for protein solubility

Hi, although most widely used, Tris-NaCl buffers may be acceptable for many but unfortunately by far not all proteins. The solubilty of a protein can be modulated by the use of the proper anions and cations. This all depends on the pH of the solution and the pI of the protein. Consult the

Re: [ccp4bb] Need suggestion for protein solubility

Hey Praveen There are wonderful advices in all the emails. Having over 8 years experience in protein purification, i still admire the way Antonio Ariza has summarized a work flow. You shall need to devise a strategy to optimzie the purification protocol. I could not resist to ask, why are not

Re: [ccp4bb] Need suggestion for protein solubility

Likely, the protein is pH sensitive. You may chain the anion and cation columns together for protein separation, then you don't need to use Imidazole. P.S. Tris pH 7.5 (RT) is ~ pH 8.2 in cold room On Sat, Dec 24, 2016 at 2:52 AM, Praveen Tripathi < tripathipraveen2...@gmail.com> wrote: >

Re: [ccp4bb] Need suggestion for protein solubility

Hi, Check pI vs pH and try different pH. Can also try room temp purification. Try lower salt conc. Is it a DNA-binding protein? Also consider adding a ligand or partner if known. Your 10% glycerol should help to improve solubility but you can also try 15% glycerol, which should also help in

Re: [ccp4bb] Need suggestion for protein solubility

AC.UK Subject: [ccp4bb] Need suggestion for protein solubility Dear all, I am graduate student working on a functional protein which i have cloned in pET-28a vector for recombinant protein production in E.coli expression system. The expressed protein is purified on Ni-NTA resins with Imidazole gradient.

Re: [ccp4bb] Need suggestion for protein solubility

Elute in batch and remove imidazole immediately :) half the time imidazole is the enemy. Artem www.harkerbio.com "From gene to sausages." Artem On Dec 24, 2016 6:04 AM, "Praveen Tripathi" wrote: > Dear all, > I am graduate student working on a functional protein

[ccp4bb] Need suggestion for protein solubility

Dear all, I am graduate student working on a functional protein which i have cloned in pET-28a vector for recombinant protein production in E.coli expression system. The expressed protein is purified on Ni-NTA resins with Imidazole gradient. Surprisingly, i am getting distinct visible white

[ccp4bb] AW: [ccp4bb] Need suggestion

as the apo-protein -By searching the internet, you can find many more tips. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von dhaval patel Gesendet: Samstag, 17. Dezember 2016 09:01 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Need suggestion Dear CCP4 users, I am

Re: [ccp4bb] Need suggestion

Hi, It's not very clear if your protein already crystallizes (maybe? otherwise why screen in a precise condition set such as yours?) and you're trying to get the ligand in... The quasi-crystalline stuff in the wells looks to me like your compound is not very soluble in the condition(s) you work

[ccp4bb] Need suggestion on classification of a protease

Dear Friends, We are doing research on membrane protease. We need to know to how to classify the protease in invitro and insilico levels. Is there is any advanced techniques or methods available, to classify the protease in insilico level? Please give suggestions on this. Waiting for your

Re: [ccp4bb] Need suggestion on classification of a protease

Hi, If you do not know anything about peptidase (protease) classification, I'd suggest you have a look first at the Merops peptidase data base: http://merops.sanger.ac.uk/ Will tell you what (type of) classes there are, for example (and based on what). Fred. Gowriishankar Raju wrote: