[ccp4bb] RES: [ccp4bb] 60 kDa contamination in Rosetta cells

I do agree with the others about what they said previously and I would not bother about the ~60kDa protein. I suppose the tag is in the 15kDa one by the amount of protein you have in the gel. I think you might be facing two different “problems”. One of your proteins (or the complex) is

Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

Sorry I got confused with the wrong labeling. But the stoichiometry issue and poor expression is still the main problem. As Tom suggested, you should try to improve the expression. Poor expression often results in impurities copurifying. Also, if the interaction is not tight, why are you

Re: [ccp4bb] 60 kDa contamination in Rosetta cells

I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern. Optimize soluble protein expression and pay attention to cell lysis. Is the sample remaining cold throughout the process? Are you using enough volume of buffer relative to solid cell pellet? Also, from the gel it

Re: [ccp4bb] Problems with refining covalently linked heme cofactor

Hi Don and Vera, Coot will handle the ligand properly without "pre-integrating" it into the PDB. Read the ligand (if it's not in the monomer library already) into aceDRG (in any form that it accepts), then in Coot, read the generated cif file -> get monomer (when positioned in the correct

Re: [ccp4bb] sphere refine radius in coot 0.9.5.1

On Fri, 2021-07-09 at 13:49 -0400, Ashok Nayak wrote: > > Is it possible to increase the radius of real space refinement Yes. Use sphere_refine with an argument, e.g. sphere_refine(8.5) You can add your own button to do this using Right Mouse on the Tool bar and selecting "Add a User-defined

Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

Hi Dilip It seems like your problem is poor stoichiometry. 37 kda protein is poorly expressed. I wouldn’t be worried too much about 60 kda protein. At-least at this stage. I might be mistaken but based on previous post, it seems like your protein was aggregated. If it is so, you

Re: [ccp4bb] Problems with refining covalently linked heme cofactor

Try Buster! Pietro Get Outlook for iOS From: CCP4 bulletin board on behalf of Vera Pfanzagl Sent: Tuesday, July 13, 2021 3:33:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Problems with refining covalently linked heme cofactor

Re: [ccp4bb] Problems with refining covalently linked heme cofactor

Dear Vera, In my experiences I had no problems to link protein atoms to a ligand in Coot. Did you check that you merged the cofactor to the protein molecule first? otherwise that could be one reason why Coot is not creating the link. BW, D On 13/07/2021 14:33, Vera Pfanzagl wrote: Hello,