[ccp4bb] Seeding and protease questions

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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

Previous message:
Subject: Re: Summary for "Anisotropic Diffraction In Refinement" question
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2009-09-15

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Subject: Re: backbone b-factor
From: Charlie Bond Charles {- dot -} Bond {- at -} UWA {- dot -} EDU {- dot -} AU
Date: 2009-09-15

Subject: Seeding and protease questions
From: Joseph Brock joeylives2ride {- at -} HOTMAIL {- dot -} COM
Date: 2009-09-15

Hi everyone,

I have two conditions that I want to try and improve by microseeding. I have been reading the literature on the subject and am a bit confused as to what exactly is the "stabilizing mother liquor" that is used in the serial dilution of the seed stock. Is this just purified protein in its stabilizing buffer? Or a mixture of this and crystallization solution? I would also like to know what the general opinion is on WHEN to add these seed stocks to crystallization drop. Is addition generally more effective upon setup or after the drops have had a chance to equilibrate?

Also, one of these conditions takes a LONG time to produce crystals (about 3 months). I understand this may be due to the slow digestion of my protein by low levels of co-purified proteases, whose slow nibbling eventually produce a truncated form that is more conducive to forming crystals. Thus, the process can be accelerated by the addition of something like pepsin to the crystallization drop. I was hoping that someone could offer advice as to what the optimal protease/concentration to use is such experiments?

Many thanks in advance for the advice and everything I have learnt from this community in the past.

Best regards,

Joe
PhD Student
Research School of Chemistry
Australian National University

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CCP4bb navigation

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

Previous message:
Subject: Re: Summary for "Anisotropic Diffraction In Refinement" question
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2009-09-15

Next message:
Subject: Re: backbone b-factor
From: Charlie Bond Charles {- dot -} Bond {- at -} UWA {- dot -} EDU {- dot -} AU
Date: 2009-09-15