Nick,
We have recently got much improved expression (both total amount and in
soluble form) of 72 kDa protein by using the EnBase Flo cell cultivation kit
from Biosilta (www.biosilta.com). The method is based on enzymatic slow
release of glucose from a starch(?)-like polymer - thus mimicking a
fed
Hi Nick,
A couple of other suggestions:
(1) Try an E. coli K strain (e.g. JM109). They're different in
glucose metabolism (JN Phue et al, 2005, Biotechnol Bioeng 90:805820)
and in my experience tend to grow more slowly - which seems to help
improve yields of large constructs.
(2) Opt
Hi Nick,
In the past, I've successfully expressed a 130 kd eukaryotic
protein in large quantities in plain BL21. I don't think size per se
is a major problem for expression although I've heard others say they
had problems with heterogeneous termination. I'd try the standard
repertoire of tech
Hi Nick,
I work with a 120kDa protein and using IPTG 50uM was crucial to get some
soluble protein. Try to reduce the amount of IPTG and temperature, increase
the induction time (24h,18°C) and volume of culture. It works fine for my
protein and may be suitable for yours as well.
Best,
Ricardo
O
+1 phenix FTW
FR
On Jan 13, 2010, at 3:28 PM, Peter Zwart wrote:
try using
phenix.reflection_file_converter data.sca
--change_of_basis="h,-k,-h-l" --sca=reindex.sca
or something like that
HTH
Peter
2010/1/13 Francis E Reyes :
Hi all
I have data integrated and scaled to P21 via denzo
Negating the DANO columns is the correct thing to do if the reindex operator
changes hkl to h,-k,-h-l which will then be put back into the asymmetric unit
with Friedel's law + symmetry
eg hkl (1,2,3) -> (1,-2,-4) ->Operator[-h,-k,-l] -> -1,+2, +4
The Friedel's law operator -h,-k,-l interchanges
Hey Nick,
Short answer is that there are no magical tricks, as you probably
already expect to hear. Seems there's a lot that you are trying already.
I have some limited experience with some 125kDa, 100kDa proteins.
1. I've had some luck with the E. coli C41, C43 strains. Especially if
your
You can use the "hkl matrix" keyword in scalepack to do this, either
on the .x files or at a push on the .sca file. See the hkl research
website to look at the manual for details.
Are you sure this reindexing does not have a negative matrix?
Johan
Dr. Johan P. Turkenburg X-ra
Hi Nick,
Some success has been reported for large soluble proteins using the C41(DE3)
and C43(DE3) *E. coli* strains (see the paper by Miroux & Walker). Also you
can try another promoter/expression system , the pBAD expression system
based on arabinose induction.
Hope this helps.
Best
--
Pasca
Hello All,
I apologize for the non-CCP4-related query. I have been working for
several weeks now trying, with limited success, to express some very
large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli.
"Limited success" means I have expressed enough soluble protein to see
Hi all
I have data integrated and scaled to P21 via denzo/scalepack. I'm concerned
about
the workflow to obtain the alternate indexing arrangement (h,k,l) ->
(h,-k,-h-l).
I was thinking .sca ( not specifying NO MERGE) -> .mtz -> reindex but the
documentation for reindex says all my DANO col
Dear Tarique,
The elbow angle in a Fab is just the angle between two pseudo-twofold axes:
one relating VL to VH and one relating CL to CH. You can find the
pseudo-twofold axes using many different least-squares alignment programs, just
pick your favorite. Then, just take the dot product of
This is not an ERROR message. It is a WARNING: Once the principal
Euler angle (or kappa) of the rotation angle relating the domains
deviates significantly from a (pseudo)dyad, the interpretation of the
kappa angle becomes somewhat obscure. The program kindly advises you
of that fact and places
Inherently you want to calculate:
1. the approximately two-fold relationship between VH and VL
2. the approximately two-fold relationship between CL and CH1
You can use many programs for that (e.g. LSQMAN) but ideally you want a
program that will report Direction Cosines for the rotation axis
A postdoctoral position in
Structural Biology of the Spliceosomal Assembly Machinery
is available at the Biocenter of the University of Wuerzburg, Germany,
within the department of Biochemistry.
The successful candidate will participate in all stages of
crystallographic structure determinati
This is just a gentle reminder that the deadline for beam time 2010 at
EMBL Hamburg is today, January 13.
With best regards,
Victor
Call for access to Synchrotron Beamline Facilities 2010
EMBL Hamburg, Germany
We announce a call for synchrotron beam time
Dear all,
I am trying to calculate elbow angle of my fab structure using a online
software developed by Robyn L. Stanfield *et. al*. but it is giving a
solution with the following errors.
WARNING: rotation matrix contains significant additional contributions
WARNING: and deviates significantly fr
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