Dear All,
Has anyone encountered problems with CCP4i/Phenix/XDS/etc on Macs with the M2
chip? I saw a post from 2020 on the M1 chip, but was curious about if there are
any issues between the M2 chip and our crystallographic software.
Thanks for any information.
Dan
Thanks for all the replies. Like I said, it eventually did upload the structure
after 20 minutes and I had no problems in analyzing the interface. I did try
two different computers, my home computer on Friday night and the lab one on
Saturday afternoon. At home it failed to load the structure,
Is the PISA server having problems? I can not seem to upload any structure.
Thanks
Dan
It has eventually loaded but has taken nearly 20minutes.
If the protein is His-tagged, load the purified protein on to a Nickel column
and place the column in a water bath above the protein's melting temperature.
Recycle 5mls of water through the column to collect the peptide and then dry
freeze or speed vac the sample to concentrate the peptide.
I
If you want a ClpP minus strain you can get it from the Keio strains.
http://cgsc.biology.yale.edu/index.php
http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp
For expression with the T7 promotor, you will need to use the λDE3
Lysogenization Kit or use a Arabinose induction plasmid instead.
Factor Xa will work depending on the exact sequence. If your sequence starts
MRS... and the start methionine is important and not removed then yes it will
work when you clone it into a Factor Xa site. If however the start sequence
starts RS... and the start methionine is actually removed, when
We have used Alphalyse (http://www.alphalyse.com/picknpost.html).
Dan
I would like to point out that HSQC could still be applied even in such a large
protein. TROSY-HSQC has been successful in improving peaks in spectra of large
protein. Typically the sample would need to be deuterated to see the full
effect of TROSY, but even a partial deuteration can improve
Producing the proteins in cell free system or bacterial expression can affect
the removal of the start methionine. Although the same rules of a small amino
acids next to the start methionine apply, different methionine aminopeptidases
tolerate certain small ones better. Depending on the which
I wish to thank everyone. I did try shifting to a higher pH and flushing 3l of
salt solution over the column which did not work. I tried 20ml 2M Urea on the
column and a stepwise shift to no urea that showed removal of the protein. I
will try binding studies to see if I did not denature the
I have a His-tagged protein which I am coexpressing with it's binding partner
to prevent proteolysis. Once on the Nickel column I can remove 80% of the
partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight.
However the last 20% is difficult to remove, even if I reload the
I am using the glutathione sepharose 4B beads from GE. I had noticed that the
capture efficiency does decrease with usage, though I have successfully
regenerated the beads at least 20 times and I am still using them.
I regenerate using 2CV of 6M guanidine after each run and every 5 runs with
THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN
June 14, 2002 The Journal of Biological Chemistry, 277, 21397-21404.
His-tagged Iron cluster that runs as a doublet. Mass-spec show they are the
same species. They concluded that the protein binds SDS in two
Have you looked at the Keio collection. Though they are not compatible with T7
promoters, you can use the λDE3 Lysogenization Kit to add the T7 polymerase.
A possible source is from Yale http://cgsc.biology.yale.edu/Auxotrophs.php
though of course there are others.
Dan
Hi all,
This maybe a simple/stupid question. I collected data at the synchrotron,
integrated and scaled the data in P222 using HKL2000, though I should of scaled
the data as P212121.
When I try to reindex using Reindex I get a message of
You are changing the symmetry of merged data are
It refolds properly according to the CD spectra but it some how manages to
hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of
sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog
that abjectly refuses to refold in either urea or guanidine,
There are a couple of papers...
Acta Cryst. (2010). F66, 346-351
Crystallization and X-ray diffraction studies of cellobiose phosphorylase from
Cellulomonas uda
The space group was originally P21. During collection the crystal moved out of
the beam (and possibly the cyrostream). Upon
I assume that the loss of the peptides that you observe for the smaller protein
is at the other terminus from the tag? If it is at the terminus where the tag
was it would suggest that removal of the tag using proteolysis is the most
likely cause. Though saying that Factor Xa/thrombin is not
According to Pierce TCEP is more tolerant of nickel and cobalt. However, TCEP
is inactivated by other metals, namely copper, magnesium, silver and zinc.
Dan
Daniel A. Bonsor,
Boston Biomedical Research Institute,
64 Grove Street,
Watertown,
MA 02472 USA
There is a nice paper
Comput Biol Chem. 2009 Dec;33(6):445-50. Epub 2009 Oct 20.
Predicting melting temperature directly from protein sequences.
Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N.
They have a list of 35 different proteins with their Tms with the references
from where they
It behaves very well. No precipitation/cloudiness during purification.
Secondary structure estimation by CD is ~50% alpha helical, 25% beta-sheet and
25% random coiled. I screened at 2, 4, 6 and 8mg/ml (8mg/ml around about
60:40% precipitation). If any other information is needed, just ask.
By size exclusion, it eluted where a 30kDa protein would be expected to elute.
As for functional assay, I am currently trying to express its binding partner,
though it shows a lot of degradation. But when I mix both proteins and run by
size exclusion it causes the peaks of its binding partner
Hello again.
At first I was not worry but maybe now I am. I have completed a structure and
submitted to the PDB. They queried my Rsym value in the highest resolution bin,
2.5-2.37A (may I dare say it 100%). I was not worried at the time as I had:
99.4% completeness
Mean(I/sdI) of 2.5
and a
Hello again!
Following my previous question, there was something wrong with the staring
model for molecular replacement. Now that is sorted, I have 8 complexes in the
ASU. After a few rounds of refinement with NCS and isotropic Bfactors, both the
Rfactor and Rfree get stuck at 30% and 36%,
Hello again...
I have a 2.7A resolution data set. Spacegroup P212121 as suggested by Pointless
and best space group when run through Phaser. Unit cell is
110.02x160.49x186.55. Mathew's suggest 11 complexes in ASU. Both Molrep and
Phaser can only find 6. This seems to be a typical observation
How do I convert the B-factors from my final structure to full B factors if I
did not use TLS refinement? I have been refining isotropic B-factors. Do I
switch to overall B-factor refinement, do something else, or have I missed the
point altogether?
It may be a dumb question but best to be
I am trying to calculate the contact surface area of a loop. Using ArealMol
I only get the overall contact surface area per residue. Is there any way to
get it per atom or does anyone know of a program (online/software) which
will perform this task.
Thanks in advance
Dan
Daniel A. Bonsor,
Sorry I should of made this clearer in my original post. Thanks anyway to
people who have responded thus far.
I am trying to calculate the buried surface area of a loop which folds from
a disordered to an ordered state.
I am looking for a program that will allow me to calculate;
(1) the buried
My first time posting to the CCP4BB board and I am very sure it will not be
my last. I have a general and specific question concerning molecular
replacements of protein-protein complexes. I have a good dataset (2.5A, 98%
complete), which has been integrated and scaled. Pointless suggested the
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