[ccp4bb] Ligand Refinement

Hello Everyone, I am attempting to fit NAD to a 2.1 A lactate dehydrogenase structure I've built and I'm running into several problems: 1. I loaded NAD from the COOT monomer library and was able to fit roughly 2/3 of it into the density using the real space refine zone function but the nicotinami

Re: [ccp4bb] ligand modelling

Hello, I suggest modelling the ligand as multiple conformations and also check if some of the conformations are coupled with the surrounding environment (side chains, waters, metals, etc.). Then, once you defined constrained groups, refine occupancies and B-factors. In my experience this work

Re: [ccp4bb] ligand modelling

On Tue, May 11, 2010 at 8:07 PM, intekhab alam wrote: > I solved one structure of a viral RNA polymerase in complex with some > ligands at 2.6 A resolution. The ploymerase structure has 3 monomers in the > asymmetric unit and in all the 3 monomers i found |Fo|-|Fc| as well as > 2Fo|-|Fc| map for

[ccp4bb] ligand modelling

Hi All I solved one structure of a viral RNA polymerase in complex with some ligands at 2.6 A resolution. The ploymerase structure has 3 monomers in the asymmetric unit and in all the 3 monomers i found |Fo|-|Fc| as well as 2Fo|-|Fc| map for the ligand. I tried to model my ligand there and after p

Re: [ccp4bb] Ion exchange protein lost

Hi Megha, how about your protein is stuck to the top of your column ? How does your pressure look like before injection and after injection ? What makes you believe that you have successfully refolded your protein ? Just some thoughts, Jürgen On May 11, 2010, at 10:53 PM, megha goyal wrote: >

[ccp4bb] Ion exchange protein lost

Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load i

Re: [ccp4bb] For 24 well Netxal plates (non ccp4 question)

Hi Theirry, Thank you very much. I am hoping for that and that is why tried the very effective ccp4bb. I will prepare a summary of the suggestions/alternatives. kind regards, Mathews From: Fischmann, Thierry [thierry.fischm...@spcorp.com] Sent: Tuesda

[ccp4bb] For 24 well Netxal plates (non ccp4 question)

Dear All, Qiagen has stopped selling the 24 well Netxal trays and changed to 15 well trays (almost same price) that doesn't fit with any screens. Do you know of some source for the 24 well screw cap trays? Thank you very much for your help, Mathews

Re: [ccp4bb] How could I Extract Iron from Protein?

I would go for DTPA and acid pH. Better than dialysis, I would use IEX and flush le bound protein with a buffer with as low a pH as possible with 10-100 mM DTPA, then wash with no DTPA, and elute le protein with clean (e.g. treated with Chelex) salt. HTH, Pr. Nadir T. Mrabet Structural& Mole

Re: [ccp4bb] control of nucleation

Hi Zq Deng No-one has mentioned microseeding into random screens, the so-called Microseeding Matrix Screening (MMS) method , or just "matrix seeding". This is exactly the kind of situation where the method works really well. You will very often get many new leads, as well as bigger crystal

Re: [ccp4bb] How could I Extract Iron from Protein?

Since you have only Asp and His ligands coordinating the Fe ion, dialysis against, say, 0.1 M Citrate at pH 5 will do the trick. Citrate will chelate the Fe(III) (if the protein has Fe(II) it will be oxidized during dialysis due to air oxygen) avoiding precipitation of Fe(OH)3 and the acid will pro

Re: [ccp4bb] How could I Extract Iron from Protein?

I would try dialyzing against a solution with 1,10-phenanthroline, 1,10-phenanthroline-2-carboxylate, or pyridine-2,6-dicarboxylate. Removal of metals from proteins is often not just dissociative, but requires the associative interaction of a chelating agent. Which one works is often empirical.

[ccp4bb] How could I Extract Iron from Protein?

Dear all,   I forgot to mention that the crystal grows in PEG3,350, Bis-Tris, and NaCl from Hampton Research. Best,   Vinson Liang

[ccp4bb] How could I Extract Iron from Protein?

Dear all,   I have a protein which binds iron with two D and two H as active site. I have tried to extract the iron by dialysis. First, 20mM tris, pH7.5, 150mM NaCl, 20EDTA, 10mM Na2S2O3, O/N. Then, followed by dialysis with 20mM tris, pH7.5, 150mM NaCl, 1EDTA O/N to remove the EDTA and Na2S2O

Re: [ccp4bb] Micro-g Crystal Growth and the literature

Dear Colleagues, Thankyou, James, for a most interesting and extensive summary. There is not much more to add. In my view microgravity has provided benchmarks for protein crystal perfection against which earth based crystal growth results can be compared. Also the general principles of crystal gro

[ccp4bb] Contact betwen multiple chains

Dear all, I like to calculate contact between one target chain, and 6 other different chains in a complex model at a time. Could you tell me how to do this? also, what is the different between contact and Ncontact programs in ccp4? Thanks in advance. cheers, V. Murugan