Hi,
You may find helpful suggestions in Kiyoshi Nagai's papers (mid 80ies) who did
this with haemoglobin (I could be wrong but I think he was the first to do
this).
Cheers,
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Hi all,
I have to merge several datasets from different crystals because the
crystals suffer from severe radiation damage.
I read some previous posts and follow the protocol as described,
1) individually process the data;
2) scaleit to compare the Rfactors between different datasets;
3)
All this is best done from the GUI - pointless will sort out batch numbers,
check indexing etc..
But you still have to identify any rogue batches, and decide on when to
jettison the data st..
The Sigma level is related to the number of observations of each reflection, so
this will decrease as
Thank you, Eleanor!
I'll try pointless. In my case, the Sigma increase as i merge all the
five data while I/sigma decrease, which is opposite to what you
describe. Is this abnormal?
Fengyun
Quoting Eleanor Dodson eleanor.dod...@york.ac.uk:
All this is best done from the GUI - pointless
Hi - SSM algorithm at PDBeFold will do this sort of thing
http://www.ebi.ac.uk/msd-srv/ssm/
I wrote a tutorial for multiple alignment to go with a Phaser story
http://www.ebi.ac.uk/pdbe-apps/quips?story=Phaserauxpage=MultiplePDBeFoldminitutorial
The last page of this tells you where to get
Dear All,
We're very pleased to announce the release of the latest version of the CCP4
Software Suite. Version 6.3.0 (Settle) is now available from the CCP4 download
website:
http://www.ccp4.ac.uk/download.php
The release is available for Linux, Mac OSX and Windows platforms, and delivers
a
BTW, a l-o-n-g time ago, I worked on a project with crystals that only grew in
the cold room. BUT... we found out that the crystals could in fact be
transferred to a regular lab under condition that you warmed them up very
slowly. So I would harvest the crystals into capillaries (this was
The Beth Israel Deaconess Medical Center X-ray Core facility in the Longwood
Medical Area of Boston, MA would like to donate the following surplus equipment
ARI Art Robbins Instruments Robbins Scientific Hydra 96 in fair condition
A set of Yale mirrors in good condition
Please see our website
Dear ALL,
We are planning to co-express two proteins in E.coli.
Could anyone suggest a good dual set plasmid or a proper insertion
sequence between two genes, including the Shine-Dalgarno sequence?
Thank you very much and have a nice summer.
Jerry McCully
Hi,
I have a crystal with a large unit cell in P21221 (a=134 b=151 c=276) and I'm
wondering if I have a problem with overlaps. I have a native dataset, and am
trying to get phases. I've collected a Pt soak data set on our home source
with a 0.5˚ oscillation angle, but the anomalous signal
I've had success with pet-Duet.
http://ecoliwiki.net/colipedia/index.php/pETDuet-1
Jason.
--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland 1142
New Zealand
ph: +64 9
I have been using the Duet system from Novagen (or whatever it is called these
days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins
did not work in either vector. Either, one protein expressed or the other. I
played around with the promotors (they are both T7) by
I was able to express a heme protein by inducing and expressing at
room temperature and using a promoter weaker than T7 (can't remember
the exact one right now). The key was to slow down the rate of protein
production to allow heme incorporation. You might try using less IPTG
too.
Ho Leung Ng
Incomplete heme-incorporation in heme proteins expressed in E. coli. is a
common issue. Check out the following paper for an easy method, which, in
our experience, has solved all our issues regarding this problem. This has
worked really well for many different heme-binding proteins (Cys / His
I have been using the Duet system from Novagen (or whatever it is called
these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of
my proteins did not work in either vector. Either, one protein expressed
or the other. I played around with the promotors (they are both T7) by
The cell predictions look like they're overlapping but the spots are not. At
first glance it looks like the unit cell is incorrect and is too large.
You seem to have intense spots mixed in with weak spots at the same
resolution. Smells like multiple unit cells / cracked crystal (which if
Hi,
The autoindexing picks this unit cell pretty much unambiguously, and the
profiles look reasonable. These are crystals of a very large heterodimer (2177
residues), and this unit cell would have 2 heterodimers and 56% solvent, which
seems reasonable. Scaling and merging produce reasonable
Hi,
This is what I thought when collecting the data - the spots did not look to be
overlapping. I have actually got 4 datasets (native, mercury, iodide and
platinum soaks) and they all index as the same spacegroup and unit cell (the Pt
soak being slightly larger unit cell). This is of a
grep SPOT_RANGE IDXREF.LP should provide you information about that. No idea
what the default would be.
How about pointless ?
Something else which might buy you a bit of signal is
NUMBER_OF_PROFILE_GRID_POINTS_ALONG_ALPHA/BETA=13
NUMBER_OF_PROFILE_GRID_POINTS_ALONG_GAMMA=13
The default for
From the statistics you posted, it seems like the integration went quite
reasonably. There is a slight undercompleteness in the high resolution bin (82%
is a bit on the low end but since this is for phasing I'd expect a traceable
map in light of this).
Do the diffraction images indicate very
Hi,
Ok, IDXREF.LP shows that it was only using 1-262. I tried running COLSPOT and
IDXREF again, and it picks the same unit cell.
Pointless picks Pmmm and picks 2 definite screw axes, and one possible (p
~0.5), so either P22121 or P212121.
I did change the number of grid points to 13 on my
I'd run
INTEGRATE(REFINE)=CELL
CORRECT(REFINE)=CELL
and fixing your distance first. Then once XDS is done copy GXPARM.XDS to
XPARM.XDS and enter those refined values into your XDS.INP script. Once you
have a stable cell you can refine the distance and later fix that one. Moving
distance is a
Hi Jason,
don't really think that the overall scaling stats look very good. Even for such
a long unit cell, we have plenty of in-house data (even with a smaller
detector) with much lower Rmerge, typically below 0.15. Possibly monoclinic
with beta close to 90deg? This might also explain the
I second Jürgen's suggestion of fixing the distance -- this is often quite
helpful when dealing with difficult datasets, at least in my experience
And this goes without saying, but also double check your beamcenter and try
masking the beamstop (using UNTRUSTED_ELLIPSE) if you haven't done so
I fully agree with Dima. We are able to co-express and purify two interacting
partners using pET28 and pET21 in E. coli. Some related references are :
J. Mol. Biol. (2011) 405,49–64
J. Biol. Chem. (2006) 281, 26491–26500
J Struct Biol. (2011) 175(2):159-70
Biswajit
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