Dear theresa
xia2 does a great job so it can be used as your reference when processing your
data manually, either for learning or improvement. When comparing different
processing programs and protocols try to judge data quality by some objectice
criteria like HA site identification, anomalous
Sorry for self promotion but you might also consider the HILIDE method which is
very similar to bicelle and sponge, only having fully solubilized
protein:lipid:detergent complexes as input sample and therefore being fully
compatible with regular liquid handling robotics.
See Gourdon P et al
Another good lesson here:
2.
The SAXS solution structure of RF1 differs from its crystal structure and is
similar to its ribosome bound cryo-EM structure.
Vestergaard B, Sanyal S, Roessle M, Mora L, Buckingham RH, Kastrup JS, Gajhede
M, Svergun DI, Ehrenberg M.
Mol Cell. 2005 Dec
But first of all: try to add a synchrotron to the crystals
Poul
On 26/01/2012, at 16.48, Katherine Sippel wrote:
Might I suggest consulting the CCP4 user community wiki on the topic:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality
Good luck,
We chewed pills with EtBr as kids in school to see if we brushed our teeth well
- red colour on the edges, bad boy
Poul
On 01/10/2011, at 19.12, Jacob Keller j-kell...@fsm.northwestern.edu wrote:
I actually looked at an EtBr MSDS a while ago, and was shocked at how
benign it was. I also
Yes - add glycerol to the reservoir -
15% glycerol has a huge influence on the vapour diffusion gradients
Poul
On 09/09/2011, at 13.22, anita p crystals...@gmail.com wrote:
Dear Crystallographers,
I have set hanging drop trays with 2ul of protein and 2 ul of resorvior
solution. I have
Calmodulin
Poul
On 23/08/2011, at 16.55, Pete Meyer wrote:
We know one protein can interact with different partners by different
domains or different parts. Is there a protein that it could interact
with different proteins by the same part (maybe the same part but in
different
Deng,
You need a good synchrotron source with a low point spread detector, but not
least a careful consideration in crystal mounting so that your crystal rotates
apprx around the long axis during data collection to optimise the separation of
reflections on the detector
besides checking that
Dear Wandu,
It might be smeared density because it is missing from your phasing model, so
no reason to reinvent the wheel with MD and Phaser. You see electron density
features for the domain and you know its basic structure. You can place the
domain manually - it would take you five minutes
Dear Art,
It seems twisted, indeed.
First of all you need to verify that your protein is at all in a good
state (you seem to imply that it is not with your remark on protein
concentration). Basic biophyscial characterisation for stability and
structural homogeniety is required to reach proper
Dear harvey
Consider your purification and preparation protocols - is the sample pure and
homogenous?
Poul
On 21/02/2011, at 03.29, Harvey Rodriguez h.rodriguez.x...@gmail.com wrote:
Dear CCP4BBers,
Recently I got a crystal which appeared in the condition 2.1M DL-Malic Acid,
pH 7.0. The
I agree with Tassos - and don't deny the power of evolution. Choice of
journals, submissions, peer review, editorial assistance and citations are all
in our hands - publishing has become what we have made it. We can argue that we
want it differently (and to stay in the evolution terms: I also
Dear dengzq1987 (Gmail Research Institute?)
You could use ammonium acetate as the solubilizing salt. It evaporates as
ammonia and acetic acid, so that the ionic strength is gradually reduced by
vapor diffusion. You would then have e.g. PEG in the reservoir and added to the
drop, and you could
[1] the signal from Ta6Br12 is enormous and one will typically focus on low
resolution (below 7 Å) so radiation sensitivity can be handled by a fairly low
dose data collection
We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth JP
et al. 2007) and found that although we
Two separate crystals, but very similar data collection strategies
P
On 04/10/2010, at 21.48, Jacob Keller wrote:
- Original Message - From: Poul Nissen p...@mb.au.dk
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, October 04, 2010 2:21 PM
Subject: Re: [ccp4bb] Radiation damage
Try a lower symmetry, e.g. P21 or P1 with one or two octamers in the asymmetric
unit (well, unit cell for P1), but you already know your packing so no problem
solving it.
It can sometimes be very subtle and your model refinement will be the most
sensitive test for the correct space group - so
Also road signs can be cleverly replaced
inline: attachment.jpeginline: attachment.jpeg
On 15/07/2010, at 16.40, Phoebe Rice wrote:
What would be wrong with WORDS? They were such a clever
invention. I can tell the difference between colors, but it
takes a second step to figure out what they
Dear Pascal,
There can be a number of reasons for this. Maybe fos-cholines are not very well
suited for your membrane proteins of interest? - have you checked for activity
or aggregation and compared to other detergents? If the detergent is optimal
you may consider moving/extending the tag or
You mention that your Rsym is 0.6 - this seems outrageously high (except if the
0.6 is just for your outer resolution bin). If 0.6 is indeed the overall Rsym
you have a basic problem of unit cell and space group assignment to reconsider.
Check if your processing accounts for all spots and check
Try first to give it a shot and see if you get crystals without too much hassle
and expense. I would recommend a size exclusion chromatography step after
refolding and from that collect a uniform sample for crystallization.
Poul
On 12/07/2010, at 13.39, meindert lamers wrote:
Dear all,
I
collection around absorption edges.
You cannot do crystallography and get and evaluate proper results without
studying the basic theory and practice - crystallography is science after all,
not black-box analysis.
Poul
Poul Nissen, professor
Whether the shiny precipitate can be redissolved or not is an important
observation. If not: bad. If yes: good
Also one can check if the shiny precipitate can be used for streak-seeding in
new drops. If not: bad/neutral. It yes: good.
A bad shiny precipitate also often forms a large connected
If you are down to a scale where you consider a single water molecule
I would say that the volume is not a very useful parameter. I woudl
rather ask if it makes chemical sense to place a water molecule, and
if the structure is determined at a sufficient level of precision that
you can
I very much agree - refinement will tell you if the high-res data make
sense. Another very good test is the Wilson plot - it should look
straight and reasonable. Inflated I/sigI values will not escape a
strange appearance such as the WIlson plot flattening out at higher
resolution. I
EF-Tu
Poul
On 18/02/2010, at 18.49, Armando Albert de la Cruz wrote:
I am trying to overexpress a His-tagged protein of 29KDa in E.coli
(BL21-codon plus) and I end up with a highly expressed product that
of 43KDa that binds to the Ni-column. I also have nice crystals.
Does anyone have any
This could be due to biscuit contamination of your protein - did you eat cake before setting up your crystallisation drops?Alternatively it could be a Fourier ripple - a very strong 5.4 Å resolution 00l reflection which is not correctPoulBegin forwarded message:From: hayato Jumonji
Hi Peter,
Histogram matching works well for RNA structure, but be aware that
density modification in general can be very tricky at low resolution.
If by any means you can exploit averaging, e.g. multicrystal averaging
of non-isomorphous crystals, it will probably be very helpful. It will
Great that you have MR phases - it will help you identify heavy-atom
sites for phasing and perhaps even the sulphur sites will be enough.
Poul
On 11/08/2009, at 20.33, ManojSaxena wrote:
Hi all,
I am working with a protein that have 28% similar to my MR template.
I have processed data in
If there is serious doubt of a full occupancy of the ligand and it is
of importance for the interpreation it can in fact be handled and even
at lower resolution (we have done it 2.8 Å resolution for PDB 2C8K for
example) - I'm not the referee but maybe he/she is right ;-)
You need not refine
We often find results to be very different between hanging and sitting
drops (equilibration kinetics for one may be the explanation). Then
there's the good thing of hanging drops that crystals rarely stick to
the surface of the support facilitating the mounting procedure, in
particular for
..and I may add that for DMMULTI two rather nonisomorphous data
sets of the same crystal form can work like a dream, in particular at
low resolution where solvent flattening and histogram matching can be
tricky, see for example Morth et al. 2007, Nature 450, 1043 Pedersen
et al. 2007,
Hi alphar~
Check the sites you get from anomalous difference Fouriers and stick
to those as your correct HA sites. At the same time you may consider
not to refine the HA substructure on the anomalous differences but
merely using the isomorphous differences. Maybe you need to change the
Besides the K-SDS precipitation it can also be the problem of membrane
proteins generally having a tendency to aggregate when boiled with
SDS. Try to just add the Laemmli buffer and load the gel without
boiling. This is the general procedure for membrane proteins.
Poul
On 18/12/2008, at
...and in absence of TM domains and the 2D restriction of the membrane
they will probably not dimerize as free domains in solution now
suddenly gained the freedom of 3D diffusion
On 11/12/2008, at 19.03, Nathaniel Echols wrote:
On Thu, Dec 11, 2008 at 8:09 AM, Santarsiero, Bernard D.
The carry-over would be DDM, and my guess would be DDM crystals formed
upon dehydration. The DDM concentration will be huge after repeated
dilution/concentration cycles. DDM micelles will hardly pass the 50
kDa cut-off filter so imagine how much you have added!
Poul
On 31/10/2008, at
located. Poul Nissen, professor, ph.d.Centre for Membrane Pumps in Cells and Disease - PUMPKIN Centre for Structural BiologyUniversity of Aarhus,Dept. Molecular BiologyGustav Wieds Vej 10C,DK - 8000 Aarhus C,Denmarkhttp://www.mb.au.dkhttp
Often the exact concentration is not so important compared to the
ability to establish proportional read-out, ease and reproducibility
so that systematic variations and comparisons can be made. For
considerations of molar ratios of for example protein:ligand complexes
one would often
Ji,
probably the oil doesn't work and the glycerol gets you into problems
with phase separation in 2.5 M AmS
I recommend sucrose or other sugars with AmS - 20-22% (w/vol) should
do and you get no phase separation
At the same time you may need to increase the AmS considerably - often
the
We normally use AmS or glycerol.
Poul
On 07/04/2008, at 15.46, Kay Diederichs wrote:
Dear all,
a protein which we work on is available in low quantity. The only
crystallization screen we set up is completely clear, no
precipitate, nothing.
Now we would like to modify the reservoirs of
Check this paper below - a C222(1) space group (a=212, b= 300, c=575) frequently appearing as a merohedral twin P2(1) with apparent C222(1) symmetry was exactly a major problem in the H. marismortui 50S structure determination.PoulBan N, Nissen P, Hansen J, Capel M, Moore PB, Steitz TA.Placement
--
Poul Nissen, professor, ph.d.
Centre for Membrane Pumps in Cells and Disease - PUMPKIN
University of Aarhus, Dept. Molecular Biology
Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark
http://www.mb.au.dk
http://www.bioxray.dk
http
purely
with MR?
perhaps in terms of %-scattering, RMSD, Z-score, LLG, or other possibly
specific scoring values.
-bryan
--
Poul Nissen, professor, ph.d.
Centre for Membrane Pumps in Cells and Disease - PUMPKIN
University of Aarhus
Hi Yuan,
Basically all tRNA structures in pdb will contain pseudo-U, and
starting from old tRNAPhe literature you will find a lot on the
structural consequences of having pseudo-U in RNA.
Poul
On 27/04/2007, at 3.45, Yuan Lin wrote:
Dear All,
I solved a structure of a
opinion, it is better to have a
good 1.8 Å structure, than a poor 1.637 Å structure.
Are these recommendations still valid with maximum likelihood methods?
We tend to use more data, especially in terms of the Rmerge and sigma
cuttoff.
Thanks in advance,
*Shane Atwell*
--
Poul Nissen
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