Re: [ccp4bb] SOme wquestions about refining.......

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Protein-DNA complex prepartion for crystallization
From: devendra srivastava dbcrystal {- at -} GMAIL {- dot -} COM
Date: 2008-10-01

Next message:
Subject: Re: Protein-DNA complex prepartion for crystallization
From: Antony Oliver antony {- dot -} oliver {- at -} ICR {- dot -} AC {- dot -} UK
Date: 2008-10-02

Subject: Re: SOme wquestions about refining.......
From: Juergen Bosch jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2008-10-02

Hi AA,

On 1 Oct 2008, at 20:34, AA wrote:

> Hello everyone,
>
> Please forgive my ignorance about the field of crystallography. I have
> recently started to process the diffraction data of a protein with a
> ligand
> attached to it. we have the structure of this protein already known
> and also
> known with different ligands. the way I did it is as flllows:
>
> I process the images in mosflm and then integrate them using scala
> inccp4i.
> After that I do molecular replacement by phaser. and finally refine
> it using
> refmac. Am I doing it correctly?
Yes that is correct but don't stop here.
>
>
> Now since I am completely new to all this, I have not much clue
> about the
> outputs I get and if they are correct or not. What are the key
> things you
> should be looking at in the log files generated by phaser and
> refmac? Till
> now I get overall R factor of 29.32 and Rfree of 34.94.
this is a solid indication that the MR actually worked - as you would
expect as you already know the structure.
> Is this fine or can
> I improve it more?
You should next use the program Refmac and refine your molecular
replacement solution to your highest resolution dataset you have.
> If yes then what do I do to further improve the
> statistics?
As a rule of thumb multiply the highest resolution limit to which your
data was processed/collected by the factor of ten this or better is
what you are aiming for with the Rfactor, the gap between Rwork and
Rfree should not exceed 5% e.g. Rfactor =25 and Rfree<=30

> What should be the RmsBond and Rms Angle values? Since the
> structure is already solved a number of times with different
> ligands, what
> final values should i aim for to get a decent density maps in which
> I can
> fit my ligand structure.
>
> Sorry for being so naive. Any sort of information or papers or links
> would
> be very helpful.
Do you have someone at your university who you could bug directly ?
It's much easier to learn from someone with experience than to read
something.
>
Good luck !

Jürgen

>
> Thanks a lot.
>
> AA

-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Protein-DNA complex prepartion for crystallization
From: devendra srivastava dbcrystal {- at -} GMAIL {- dot -} COM
Date: 2008-10-01

Next message:
Subject: Re: Protein-DNA complex prepartion for crystallization
From: Antony Oliver antony {- dot -} oliver {- at -} ICR {- dot -} AC {- dot -} UK
Date: 2008-10-02