Re: [ccp4bb] the purification process of protein, used for crystallization

Hi Liuqing, The scheme you suggested (or heard) of using Ion-exchange at the end can be useful to separate protein with same net charge but different conformations. Ciao On Fri, Nov 17, 2017 at 1:49 PM, Gianluca Cioci wrote: > Dear All > > The ion exchange has the

Re: [ccp4bb] high TFZ score but 50% of Rfree

Do the following. 1) Perform density modification (NCS averaging and solvent flattening) 2) Run the new phases through a model building program (Buccaneer works for all resolutions, Arp/wArp works well for better than ~2.7 Å or better resolution) If this doesn’t give you a better quality model

Re: [ccp4bb] high TFZ score but 50% of Rfree

R=50% at a resolution of 2.2 Å is a lot different to 50% at, say, 3.5 Å resolution. What happens if you refine it at 3.0 or 3.5 Å ? What's the model vs sequence % identity ? Phil Jeffrey Princeton On 11/17/17 3:23 PM, Yue Li wrote: Dear all, I have several datasets (one best resolution

[ccp4bb] high TFZ score but 50% of Rfree

Dear all, I have several datasets (one best resolution reaching 2.2A), giving C2 space group, two molecules in an asymmetry unit (65.1% of solvent content).  When running MR using a template (<20% sequence identify to the target molecule), I got a solution with high TFZ (23.7) and LLG (842).

Re: [ccp4bb] the purification process of protein, used for crystallization

Dear All The ion exchange has the great advantage ,over other tecniques, to concentrate the protein. Histrap+sec is a big classic in protein purification but times it is worth considering other schemes. Why doing a SEC? It is for removing aggregates or a contaminant ? It the latter case probably

Re: [ccp4bb] the purification process of protein, used for crystallization

Hi Liuqing, I would not recommend SEC. SEC does not give that great of a separation unless your contaminant is greatly different in size. Instead of SEC, you might want to consider hydrophobic interactions chromatography (HIC). You can add your ammonium sulfate directly to your eluted protein

Re: [ccp4bb] the purification process of protein, used for crystallization

Hi Liuqing, I agree with what Pascal has already written and can add something from my experience. In one case I needed to purify a protein using gelfiltration to control the exact composition of the buffer before preparing for the ion exchange. The protein was only stable in high salt buffers.

Re: [ccp4bb] the purification process of protein, used for crystallization

Hi liuqing, It is more usual to finish with a sec because you control the composition of the final conditioning buffer of your sample and remove any aggregates. We personally favor the following sequence iMac - desalt - iex if necessary - sec Desalt meaning one of those small Pd10 like column

Re: [ccp4bb] CCP4i2 Error in wrapper

Hello, I am not sure but I had the same kind of problem using ccp4 on Linux "exotic" distribution which use python 3 by default. Check that your Linux installation has python 2.x as default python in your environement. Hope this guide a bit the trouble shooting. Nicolas Horrell,

[ccp4bb] CCP4i2 Error in wrapper

Hello CCP4bb, I'm having some trouble with my installation of CCP4i2. I keep getting a persistent error about the "Wrapper" in various programs and am not sure what I need to do to fix it. I have tried reinstalling via the package manager (64b linux) and still get the error. So far it has

[ccp4bb] the purification process of protein, used for crystallization

Hello everyone! I have listened someone suggested that, first use affinity chromatography (Ni-NTA), then use SEC (superdex200 increase), and finally used ion exchange (monoQ), to purified protein, which will be used to crystallization. My question is why the monoQ used in the finally

[ccp4bb] Crystallographer position at SARomics Biostructures

Hi, The structural biology CRO SARomics Biostructures in Lund, Sweden, has an open position for a protein crystallographer. The application deadline is 1st December. See here for details: https://www.saromics.com/About/About/Career.html Best regards Derek Logan

[ccp4bb] Final reminder - CCP4 Study Weekend 2018 - Only 3 days until Early bird deadline on 19 November 2017 (this Sunday!!)

Please note that the Early bird deadline for the forthcoming CCP4 Study Weekend in 10-12 January 2018 is 19th November 2017 i.e. this Sunday. After this date, no standard student bursaries will be available and the registration fee will increase from £240 to £290 for the final 3 weeks of

Re: [ccp4bb] Scripting for COOT

We have used it in this way... coot --pdb input.pdb ---auto input.mtz --script script.com and then, in file "script.com", you write the corresponding instructions... (set-go-to-atom-molecule 0) (set-go-to-atom-chain-residue-atom-name "B" 42 " CA ") Martin