Hi Ivan,
Did you try using a buffer other than phosphate? Also maybe a different pH can
help keeping the IgG in solution. Although papain prefers pH 6-7, it is a
fairly robust enzyme and will cleave with >20% efficiency in the range of pH4-9
(Hoover S & Kokes E ,1946, http://www.jbc.org/content
Thanks everyone,
I have really a lot to do now.
Jurgen,
yes I do separate the complex over size-exclusion column before setting
trays.
Patrick,
Thanks for the wonderful reference.
Sivaram,
Thank you.It was really nice of you to send the link of your thesis.It is a
wonderful gesture and I apprecia
Hi Pietro,
There are monomer library entries for Ca in various states of
coordination by water, entries, OC1, OC2,OC3 etc but unfortunately
these are incomplete (no distances or angles with sd's), at least in
our CCP4 installation. The entry for octahedral MG-O6, file MO6, is
complete so
Pietro,
You will need to set "Make links between: All others if...residues are
close only" in the Refmac GUI and write out a .cif file for the
Ca-ligand distance restraints. See this link:
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement#Restraining_metal_ligand_distanc
Dear all,
How do I set up a Refmac dictionary to constrain octahedrally coordinated Ca++?
Refmac5 seems to detect the potential bond between the Ca++ and the ligands:
INFO: link is found (not be used) dist= 2.229 ideal_dist= 2.320
ch:AA res: 263 GLU at:O .->ch:Ag
Again you should read the spectacular paper by Obmolova and co. where
they solved the structures of three Fab-antigen complexes using "MMS"
microseeding (seeding into random screens), starting with one hit
containing clusters of needles - which could not themselves be optimized
The paper is ava
