Hi Omi
Did you test them for diffraction? you might want to keep screening
broad for better crystals. You could try to add K/NaSCN, KI or KNO3 as
an additive (50 mM) to you protein mix and go from there
best Preben
On 8/23/13 4:56 AM, zhuqing ouyang wrote:
Dear all, I am trying to crystallize
Hi
Some people emailed me saying that the attachment did not get through.
I hope this will work.
Sorry.
D
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry
[ber...@upstate.edu]
Sent: 23 August 2013 00:01
To: ccp4bb
Subject
Hi Gloria,
You can paste your protein sequence directly into the text search box of the
RCSB PDB website (http://www.rcsb.org), and choose one of the sequence search
autosuggestions.
You can also to try the PSI-BAST search to find more distantly related
homologs: click on the Sequence option i
OK, I see my mistake. n has nothing to do with higher-order
reflections or planes at closer spacing than unit cell dimensions.
n >1 implies larger d, like the double layer mentioned by the original
poster, and those turn out to give the same structure factor as the
n=1 reflection so we only consid
Dear Community,
I have attached a short booklet written some 6 years ago during rainy evenings
to teach principle of crystal diffraction with biologist students in mind,
never used it as I don't have students, but I now believe its mission was this
thread ;-)
It uses lots of real space diffrac
Hi Everyone,
Just wanted to let everyone know that i was able to process this dataset
with XDS ( and lots help from experts !)
Thanks again
Mahesh
On Mon, Aug 19, 2013 at 5:07 PM, Petri Kursula wrote:
> I have often processed images like this with XDS. Of course, you will get
> a better qual
On Thursday, August 22, 2013 02:19:11 pm Edward A. Berry wrote:
> One thing I find confusing is the different ways in which d is used.
> In deriving Braggs law, d is often presented as a unit cell dimension,
> and "n" accounts for the higher order miller planes within the cell.
It's already been
One thing I find confusing is the different ways in which d is used. In deriving Braggs law, d is often presented as a
unit cell dimension, and "n" accounts for the higher order miller planes within the cell.
But then when you ask a student to use Braggs law to calculate the resolution of a spot
On Thu, 2013-08-22 at 14:22 -0400, Bosch, Juergen wrote:
> But when it sits and crystallizes it is cleaner - unless some
> opportunistic fungal contamination helps you trim off those nasty
> loops that you would have omitted anyhow from your model.
Jurgen,
Good point and admittedly I never consi
I thank everybody for the interesting thread. (I'm sort of a nerd; I find
this interesting.) I generally would always ignore that n in Bragg's Law
when performing calculations on data, but its presence was always looming in
the back of my head. But now that the issue arises, I find it interesting
Here is why we use the batch method.
1. We do it in the cold and do not have a working FPLC in a cold box or
room.
2. Our volumes are large and do not have an easy way to load it using an
FPLC.
3. The buffer volumes are greatly reduced using batch. This can be an
issue with some detergents.
C
But when it sits and crystallizes it is cleaner - unless some opportunistic
fungal contamination helps you trim off those nasty loops that you would have
omitted anyhow from your model.
Jürgen
P.S. I like kangaroo's in particular when served with red wine and medium rare
next to some garlic ma
On Thu, 2013-08-22 at 13:58 -0400, Bosch, Juergen wrote:
> well if we hit the timer after lysis, say via cell disruptor then I
> have my eluted protein in less than 1 hour, including 40 minutes batch
> binding.
then proceeds to wait six weeks for crystals to appear... :)
Cheers,
Ed.
PS. There
Dear Pietro,
The n in Bragg's Law is indeed most interesting for teachers and a most
delicate matter for those enquiring about it.
The diffraction grating equation, from which W L Bragg got the idea, a 'cheap
accolade' he said to have it named after him in his Scientific American
article, has
herman.schreu...@sanofi.com wrote:
Dear James,
thank you very much for this answer. I had also been wondering about it. To
clearify it for myself, and maybe for a few other bulletin board readers, I
reworked the Bragg formula to:
sin(theta) = n*Lamda / 2*d
which means that if we take n=2, for
Hi Ed,
well if we hit the timer after lysis, say via cell disruptor then I have my
eluted protein in less than 1 hour, including 40 minutes batch binding. If I'm
pressed for time I bind only 20 minutes and then can have the protein ready for
the next step within 30 minutes after lysis. Some of
On Thu, 2013-08-22 at 10:07 -0400, Bosch, Juergen wrote:
> Yes, I'm also surprised why people run gradients for the capturing
> step ?
Because we can. Joking aside, I've seen some examples where protein
eluted at relatively low imidazole and upon running the gradient there
remains some (minimal)
On Thu, 22 Aug 2013, Gloria Borgstahl wrote:
We have a protein sequence that probably contains OB folds. What is the
best way to search for the top structural homologs to this sequence in the
pdb? G
Hi Gloria,
If you expect decent sequence simnilarity to one or more proteins in the PDB,
an
Hi Gloria,
I've gotten useful results from these two threading / prediction servers:
Raptorx and Phyre (for low homology, I found the original version works better
than phyre2).
Phoebe
++
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The
Dear Gloria,
If you are searching with a protein sequence, you could also try FFAS
(ffas.burnham.org/). This will perform profile-profile alignments to do fold
recognition. Works quite well for low homology cases in particular.
HTH and best wishes,
Mohammad
--- Begin Message ---
We have a p
Instead of DALI, you might also want to try SALAMI
(http://www.zbh.uni-hamburg.de/salami) for your structure searches.
Tommy
On Aug 22, 2013, at 5:17 PM, Tim Gruene wrote:
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear G,
>
> if you only have the sequence, I suggest a BLAST search
Dear Gloria,
HHpred.
Best wishes,
Tomas
On Thu, Aug 22, 2013 at 4:14 PM, Gloria Borgstahl wrote:
> We have a protein sequence that probably contains OB folds. What is the
> best way to search for the top structural homologs to this sequence in the
> pdb? G
I am not sure if this is the best way but I always use Blastp and search
against PDB. Dali search would be more appropriate if you have a structure of
your protein.
Zhen
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gloria
Borgstahl
Sent: Thursday, August 22, 2013 11:15
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear G,
if you only have the sequence, I suggest a BLAST search
(http://blast.ncbi.nlm.nih.gov/Blast.cgi), if you do have the
coordinates, you can use the DALI server
(http://ekhidna.biocenter.helsinki.fi/dali_server/start)
Regards,
Tim
On 08/22/201
We have a protein sequence that probably contains OB folds. What is the
best way to search for the top structural homologs to this sequence in the
pdb? G
Yes, I'm also surprised why people run gradients for the capturing step ? How
often is your desired protein spread out throughout the whole gradient ? So
what's the point the of the gradient if in the end you merge the whole peak
again ?
Maybe for a pilot study to identify how much imidazole cou
I've never done the experiment, but it is easy to determine the absorption
spectra of imidazole at different pH values and see if there is an
isosbestic point - and then plot (A280 minus the Abs at the isobestic
point), vs pH, and see if the pKa is about 7.0. Imidazole is aromatic as
the free
The absorbance is due to imidazole, for sure.
We do a lot of this type of purification in my lab and we
have definitely had some low abundance proteins hide under the imidazole
gradient.
Here is how we deal with it.
We expect the proteins to elute around 100-350 mM imidazole, so we run
SDS-PAGE on
Hi Andrew,
1. Some boundaries give much worse or even unstable refinement whereas others
give perfectly reasonable results. There seem to be real sweet spots for TLS
group selections; the question is whether these relate to the mechanics of the
structure. The system is well enough determined if
Hi Robbie,
I was interested in a couple of things in your response:
1. Slightly different boundaries giving surprisingly different results.
Doesn't this suggest that the system is too poorly determined to justify using
TLS (and there is the issue that Ethan Merritt has raised m
Adding to what James wrote: I see this as follows:
Bragg's law is only a necessary but not a sufficient criterion for
occurrence of a diffraction peak if viewed as a reflection. The problem imho
comes from not considering the structure factor as the actual quantifier of
(single photon) diffraction
Dear James,
thank you very much for this answer. I had also been wondering about it. To
clearify it for myself, and maybe for a few other bulletin board readers, I
reworked the Bragg formula to:
sin(theta) = n*Lamda / 2*d
which means that if we take n=2, for the same sin(theta) d becomes twice
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