Larry Grant wrote:
I am attempting to fit NAD to a 2.1 A lactate dehydrogenase structure
I've built and I'm running into several problems:
1. I loaded NAD from the COOT monomer library and was able to fit
roughly 2/3 of it into the density using the real space refine zone
function but the
Dear ccp4bb,
We used to routinely overexpress a number of mammalian proteins to
inclusion bodies in E.coli. Lately however we get from time to time
smeared and small or no pellets after spinning down the cultures (lysed
I assume).
I have read (with some horror) previous threads pointing at pha
Hi Hannes,
We have recently had a batch of phage contamination - one of the
symptoms was exactly as you described, when pelleting the cultures, a
smeary, fluffy pellet appeared. The other symptoms we had were that
during growth, the OD would rise up until ~0.5, then drop, a foam
would develop on t
Dear Hannes,
I am afraid it can still be phage problems. There are many kinds of phages that
infect E. coli and some of them behave like you describe: not always
lysed cells but from time to time and coming and going. The phage may anyway be
present
but in its lysogenic state and only entering t
Structural Biologists (Ref SB-10)
10/05/2010
Cambridge Science Park
Astex Therapeutics is a world leader in fragment-based drug discovery.
The company has a strong commitment to X-ray crystallography - all our
projects are based on a detailed understanding of protein: ligand
interactions and th
Usual apologies for non-CCP4 question.
I have been working on two high resolution protein structures using
Shelx for refinement.
I included idealised riding C-H and mainchain amide N-H hydrogens in
the refinement. In the PDB output by Shelxl the hydrogen atom names
for valine, threonine,
We have had problems with phage contamination, either through
contaminated plasmid stocks or more sporadic infections. Even if the
cultures don't look clear, you can usually spot the contamination if
the E. coli pellets are slimy and spread all the way up the centrifuge
bottles. We have had
Dear Alice,
I don't know what the consensus about this is and I don't know your resolution.
But if you really only have hydrogens in riding positions and did not refine
them you might as well deposit the structure without hydrogens in the PDB-file.
You could also check if shelxpro deals with the
Dear Alice,
There is a very simple workaround: simply don't deposit the hydrogen
atoms (if you use the B option in SHELXPRO to prepare the deposition
this is the default anyway). SHELXL refined them as a riding model for
the xyz and B values anyway. If anyone needs them later they can easily
re
-- Forwarded message --
From: Arpit Mishra
Date: Wed, May 12, 2010 at 6:20 PM
Subject: regarding purification of DNA binding protein
To: ccp...@jiscmail.ac
hi all
i am trying to purify DNA binding protein, but i couldnt get rid of the DNA
after performing hi-trap heparin column,
Dear Arpita,
For Getting rid of DNA you can incubate the lysate with DNase and incubate
it before lysing the cells with sonication or french press (or anyother
method of your choice)
cheers
Rashmi
On Wed, May 12, 2010 at 8:53 PM, Arpit Mishra wrote:
>
>
> -- Forwarded message
Hello Arpit,
don't you use DNAse during lysation of the cells? Or do you mean that you want
to get rid of unspecific DNA and want to add some specific DNA after
purification? In that case I'd understand you don't want to use DNAse.
Tim
On Wed, May 12, 2010 at 06:23:56PM +0530, Arpit Mishra wrote:
Hi Arpit,
You can use PEI (polyethyleneimine). Adding PEI at low conc. (e.g. 0.25%)
after cell lysis should precipitate most of the DNA. Note, it is best to do
the addition step-wise at 4 deg with gentle steering over a period of time
10-15 min or so.
Ibrahim
On 5/12/10 10:20 AM, "Tim Gr
Hello all,
I would like to thread one protein sequence onto a structure I recently
solved, and has not been submitted to the pdb.
I found the swiss-model website, which is an excellent tool for inputing a
template sequence and target sequence (which is what I want to do), but it
only allows you to
Dear All
The following position is available:
Research Scientist
Texas A&M University
Microscopy & Imaging Center
The MIC is looking to fill a permanent position (competitive salary and
comprehensive benefit package included) with an energetic and passionate
electron microscopist well versed in
Kelly,
This can be done with the Swiss-PDBviewer itself.
Download the viewer if you don't have it.
load your pdb ( File > Open PDB File... )
load the sequence you want to thread (fasta format) ( SwissModel >
Load Raw Sequence from Amino Acids...)
then thread with ( Fit > Fit Raw Sequence)
Good L
Kelly,
You can do it with swiss-model, just have to use the alignment mode. I will
ask for a pdb or RCSB code. Also, in the past I have found Jigsaw 3D model
server to be a comparable tool.
Scott
On Wed, May 12, 2010 at 10:10 AM, Kelly Daughtry wrote:
> Hello all,
> I would like to thread on
The following positions are available at the Institute of Biochemistry,
University of Lübeck, Germany:
PhD students and postdoctoral fellows in (structural) virology and
synthetic organic chemistry
1. Postdoctoral fellow in crystallography/structural virology (initially
limited to 2 years; e
Hi,
Which free online servers are best for calculation of non covalent
interactions present in several pdb structures?
Thanks for any insights provided by the community members...
Cheers
GM
Like Juergen suggested, your protein is most likely stuck to the column,
either because it never was really was refolded or because it doesn't
like to be at pH 4.5. Do you really need such a low pH to have it bind
to source 15S. Check the pI of the protein and perhaps some different pH
buffer o
Human gcsf has a pI ~ 6.
As Ursula suggests you might be better off with anion exchange.
There are protocols that show you can both refold and purify onto such
column type.
Nadir
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medic
Thanks for all the suggestions.
I was able to use swiss-pdb viewer to accomplish my goal within 2 minutes of
installing!
Very useful software in general! I didn't realize all of the tools present.
Thank you everyone!
Kelly
***
Kelly Daughtry
P
(I noticed these words of wisdom did not make it to the thread. Hereby
belatedly corrected.)
The relevant TLI here is ROI: for the money to put one crystallization
plate in space, you can employ enough postdocs probably to solve all
membrane proteins in the human genome terrestrially 3 times o
You could try LPC CSU Server:
http://bip.weizmann.ac.il/oca-bin/lpccsu
-Gyan
On Wed, May 12, 2010 at 1:02 PM, gauri misra wrote:
> Hi,
> Which free online servers are best for calculation of non covalent
> interactions present in several pdb structures?
> Thanks for any insights provided by
Tillmann,
I've added a jiffy script to synthesize pseudo-precession photos from a
rotation dataset, to the latest PHENIX package. We build this package
nightly, so any PHENIX bundle with a version number greater than
"dev-402" will workhowever I see that dev-402 is not yet on the
public
Dear All,
Just a reminder that May 15, 2010 is the deadline for the July-August
2010 Rapid Access Proposal cycle for PX beamtime at the ALS. The BCSB
beamlines (5.0.1,5.0.2, 5.0.3, 8.2.1 & 8.2.2) can all be operated
remotely and are all equipped with ADSC Q315(R) detectors. Beamlines
8.2.2, 8.2.1
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