Hi,
from my experience with minimal media expression you're probably
better off starting with a good, thick rich media overnight growth,
and then diluting that out 1:20 – 1:50 into the M9.
Richard
--
Richard P. Grant
School of Molecular & Microbial Biosciences University of Sydney
Hello everybody,
Sorry for an offtopic question. I am trying to express a protein in M9
minimal media for Selenomet incorporation. When grown in LB this protein
expressed very well and got good crystals. Diffraction was upto 2 A. I am
having a hard time expressing the same protein in Minimal me
Hi Nick, it's not obvious to me this is a single lattice, hence the "many"
spots. But it's not protein, don't worry about that.
phx.
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Noinaj
Sent: 19 April 2007 00:19
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] prot
Hi,
In my opinion this is clearly a twinned crystal of some sort of a small
molecule. If you have a small ice crystal on top of a completely
non-diffractive protein crystal, sometimes the pattern may also look like
this - but I would bet this is just 'salt'.
Artem
_
From: CCP4
We are looking for a new member for our group...the ad follows.
-Dirk Bussiere
Structural Chemistry Group, Structure-Based Drug Design & Discovery
Novartis Institutes for BioMedical Research, Emeryville, California
Use your scientific expertise to aid us in our structure-based drug desi
Hi,
i collected a diffraction pattern today of a protein crystal, hoping to answer
the question, is it a protein or salt? The crystals appear as typical proteins
crystals (from my experience), forming both plates and rods, easily crushed
into millions of tiny crystals, and are bouyant. unfor
My previous posting did not get answer, it might be a bit confusing. My
questions is if I want to conbine phases from three different sources, 2
MAD data sets and a partial model, can I run SIGMAA twice? Combining MAD
phases first, and then add in the model phases to the pre-combined MAD
phases
You would probably be better off using the CentOS5-supplied python, tck,
tk, blt, and so forth (and if they aren't supplied, then consider a linux
distro that provides these things).
Also, if you compile your own ccp4, you can include the latest patches and
direct it to use whatever versions of th
Hello,
I have been running into issues with the tcl/tk and python bundles currently
distributed with ccp4-6.0.2 (x86 system running Centos5).
1. The tcl/tk/blt package for Linux now extracts into a ./tcltkblt
subdirectory. Apart from making upgrades slightly easier, this does not seem to
make v
Mike,
This is a complicated question :) The floppy bits can contribute to
packing in several non-obvious ways.
I may be able to help you get a better crystal form, if you're willing to
share the specifics of the protein (which will be kept confidential of
course). I've developed new procedures fo
Eva,
I have used CNS to refine several low resolution structures. I have
even developed several tools specifically for low-resolution refinement
in CNS (see http://xray.utmb.edu/PMB/index.html#BPATCH). However, I
agree with the advice given by most of my colleagues, refine isotropic
B-factors in
Here's an interesting paper on "The Genesis of Twinned Crystals" (from
1945! - it's even printed on simulated aged paper and if you look
closely the "aging" has an unrealistic plane-group pattern!):
http://www.minsocam.org/msa/collectors_corner/arc/twinorig.htm
It's by the eminent crystallograp
On Wednesday 18 April 2007 07:40, Phil Jeffrey wrote:
> Harry M. Greenblatt wrote:
>
> > You should be refining an overall temperature factor at that
> > resolution. It's one of the choices in the list, instead of "isotropic".
>
> I disagree with this. At that (3.2 Angstrom) resolution I've
Thank you Roberto, I saw that line in the log file, too. So it is as I
feared: Refmac cannot be stopped from refining B-factors ;-) Maybe I'll use
CNS...
Eva
2007/4/18, Roberto Steiner < [EMAIL PROTECTED]>:
On 18 Apr 2007, at 14:39, Eva Kirchner wrote:
(But I'm still curious about the B
Harry M. Greenblatt wrote:
You should be refining an overall temperature factor at that
resolution. It's one of the choices in the list, instead of "isotropic".
I disagree with this. At that (3.2 Angstrom) resolution I've often
found than a tightly restrained individual B-factor refinemen
Greetings,
I've taken a protein (structure known) with some disordered termini and
created a new truncated construct that lacks these disordered bits (approx.
5% by mass of the total protein).
To my delight, the new construct crystallizes much more readily than the
full-length one (more hits in
On 18 Apr 2007, at 14:39, Eva Kirchner wrote:
(But I'm still curious about the B-factor refinement when there is
no "REFI BREF ISOT" in the com-file...)
Eva,
Refmac internal default is REFI BREF ISOT
that's why even if you remove the above line from the com file (or
deselect that op
Dear Eva and Harry and others,
I am not sure that an overall B-factor is the best solution for a 3.2 Å
structure. In general, the true B-factors will vary a lot for different parts
of the protein and poor diffracting proteins often have parts which are
partially or completely disordered. An ov
Thank you both, Mischa and Harry, I'll try overall B refinement. But I'm
starting to think that my Refmac is somewhat crappy, I tried this before,
and then all the B-factors were exactly the same. Now, I tried it again and
they are not... Something's still wrong here.
(But I'm still curious about
This is not definitive, but in a similar case - P21 with a~c, we could
detect twinning from the moment plot in TRUNCATE with 15% of data.. (ie
run SCALA and TRUNCATE option - scales poorly determined, and I think we
fixed them to unity..)
Eleanor
That meant you could test the crystals at ho
Thanks to everyone for all the great suggestions.
Regarding which mtz to use as a starting point, advice was to either re-run
scala selecting the option to output unmerged reflections, "scaled, unmerged &
no outliers" under "Customize scala process" in ccp4i (Miguel Ortiz Lombardía),
or to g
Can you (or anybody else) expand upon this -- what would be a
recommended screening protocol for twinning? Collect X degrees of data,
scale, merge, run mmtbx.xtriage? How many degrees of data will it take
to give a reliable estimate, and does this depend upon crystal
orientation, SG, merohedral v
BS"D
Dear Eva,
You should be refining an overall temperature factor at that
resolution. It's one of the choices in the list, instead of
"isotropic".
Hi,
I have a little problem with B-factor refinement. I'm using the
CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tri
Hi,
I have a little problem with B-factor refinement. I'm using the CCP4i
interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as
well, it doesn't make a big difference for this problem), and a current
Rfree of 30.4%.
Refmac refines the B-factors so that they are nearly the same
I seem to recall a discussion at some Study Weekend where someone
reported a needle crystal in which the twin fraction appeared to vary
continuously from 0 at one end of the needle to 0.5 at the other! You
just had to make sure you picked the right end to collect the data!
-- Ian
> -Origina
This is a very common thing - twinning is a sort of accidental overlap
of crystal fragments and thus will be different for diferent
"crystals". ( Should a twinned object be called a crystal? Discuss)
More serious - it is probably different for different parts of the data
collection if your cr
Hi Mark,
thus far, in the many cases where we had merohedral twinning, the twin
fraction varied usually a lot, ranging from ~0.1-0.5 for the same
crystallization conditions. So, it's a good idea to screen a couple of
crystals and check their twin fractions (estimating the true twin
fraction i
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