We've been using the S219V mutant for cleaving out tags. We usually do our
cleavage reactions in an overnight dialysis after a Ni-column into 50mM Tris,
100-200 mM NaCl, 5 mM BME at 4C. I've never found problems with losing any
protein in the reaction and recover usually >90% of the protein afte
Hi to all on ccp4bb:
What better day to announce the availability of a breakthrough technique
in macromolecular crystallography?
Given recent discussion and in particular James Holton's suggestion that
the problem of disordered sidechains is a problem akin to the difficulty
of describing dark mat
Dear All,
just in time for the heated discussion of missing density, high B-factors, and
split conformations I have a paper in the most recent Phys Rev Letters that
provides a rational explanation for the observation of split conformations when
the equilibrium density becomes weak.
The first p
Ian,
I think it's amazing that we can program computers to resolve a < b < c
but it would be "a major undertaking" to store the matrix transformations
for 22121 to 21212 and reindex a cell to a standard setting. I was also
told that I was lazy to not reindex to the standard setting when I was a
gr
On 31/03/2011 23:43, Zbyszek Otwinowski wrote:
Regarding the closing statement about the best solution to poorly
ordered side chains:
I described in the previous e-mail the probabilistic interpretation of
B-factors. In the case of very high uncertainty = poorly ordered side
chains, I prefer to d
This can be very hard to do because quite a few proteases are promiscuous
and will cut substrates solely based on masking of the polypeptide within
the structure of the protein. Typically these proteases will not stop
cutting at a single nick - they often proceed until they can't 'dig into' a
burie
Hi Bernhard,
I realized this when comparing the functions yesterday. Exactly like what
you said, when a user interrupts the process, what he/she wants at the
moment is most likely to be going back a few residues instead of starting
all over again. I am quite happy to have both interruptible an
Try fitting a xylitol molecule in it but watch out for the distortions
caused by proximity to symmetry axis.
Artem
On Thu, Mar 31, 2011 at 1:16 PM, Shu XU wrote:
> Hi, there.
> I'm refining a 1.76 A structure. The r-work stuck at 21%, rfree is 24%
> after adding waters.
> But there is something
I agree with Zbyszek with the modeling of side chains and stress the
following points:
1) It drives me nuts when I find that PDB is missing atoms from side
chains. This requires me to rebuild them to get any use out of the PDB
such as relevant surface renderings or electropotential plots. I am
On Thu, Mar 31, 2011 at 10:43 PM, James Holton wrote:
> I have the 2002 edition, and indeed it only contains space group
> numbers up to 230. The page numbers quoted by Ian contain space group
> numbers 17 and 18.
You need to distinguish the 'IT space group number' which indeed goes
up to 230 (i
Regarding the closing statement about the best solution to poorly
ordered side chains:
I described in the previous e-mail the probabilistic interpretation of
B-factors. In the case of very high uncertainty = poorly ordered side
chains, I prefer to deposit the conformer representing maximum a
Dear Ed,
Thankyou for this and apologies for late reply.
If one has chemical evidence for the presence of residues but these residues
are disordered I find the delete atoms option disagreeable. Such a static
disorder situation should be described by a high atomic displacement parameter,
in my vi
Hi All,
Notwithstanding the stimulating discussion about the B-factor, I'd like
to chime in with my $0.02 on the original question of to build or not to
build and what are the rules and standards... and sorry for the lengthy
e-mail - I was trying to respond to several comments at once.
I thought t
Pat,
at least give it a try with the one sweep approach.
We have collected plenty of 360deg data sets on a Rigaku system which requires
two omega sweeps at phi 0 and 180 deg. These data sets are for in-house
phasing. We haven't seen big issues with running XDS over these images as one
continuous
Dale Tronrud wrote:
While what you say here is quite true and is useful for us to
remember, your list is quite short. I can add another
3) The systematic error introduced by assuming full occupancy for all
sites.
You are right that structural heterogeneity is an additional factor.
Se-Met
We've just collected a number of inverse beam data sets. It turns out the
crystals showed little radiation damage, so we have a lot of data: 2 x 360 deg
for each crystal, broken up into 30 deg wedges. The collection order went like
this: 0-30 deg, 180-210, 30-60, 210-240, etc.
Now, assuming no
I once cleaved a GST tag on the resin using TEV by rocking overnight at 4 C.
I would say it is 100% cutting judging from the gel. One thing to add is
that the protein bound so tight to the beads that cutting tag is the only
way to elute it except by SDS. I haven't had any trouble with TEV and
throm
On 3/31/2011 12:52 PM, Jacob Keller wrote:
The only advantage of a large, positive, number is that it would create
bugs that are more subtle.
Although most of the users on this BB probably know more about the
software coding, I am surprised that bugs--even subtle ones--would be
introduced by re
I have the 2002 edition, and indeed it only contains space group
numbers up to 230. The page numbers quoted by Ian contain space group
numbers 17 and 18.
Although I am all for program authors building in support for the
"screwy orthorhombics" (as I call them), I should admit that my
fuddy-duddy s
Totally agree with Chun.
We are using a His tagged S219V construct that's very similary to the one
described in the Waugh paper. To my experience, agitation, 37C incubation, low
salt buffer, (etc.?), should all be avoided when using TEV. When using
relatively large amount of enzyme(0.1~0.2mg/m
There are no 'new' conventions to keep up with: recent editions of the
old volume 1 or new A do not disagree on the question of the unit cell
conventions (except for minor details which don't affect the majority
of the common space groups), where by "recent" I mean "going back ~ 70
years". So it's
On another note, we have moved from baculovirus to stable poly or
monoclonal Tn5 cell lines using the pIB/V5-HIS vector. It removes all
concerns about titering and viral propagation, and for most of our
proteins, the expression level is the same or better. We secrete all
our proteins, so the stab
Properly made TEV protease should work with or without rocking. We have done QC
of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used
on hundreds of proteins. The most common cause of incomplete digestion is the
insolubility of target protein or bad construct design. Sh
> The only advantage of a large, positive, number is that it would create
> bugs that are more subtle.
Although most of the users on this BB probably know more about the
software coding, I am surprised that bugs--even subtle ones--would be
introduced by residues flagged with 0 occupancy and b-fact
As many have mentioned, there is no need to titer baculoviruses for protein
expression purpose. Plaque assay or other titering methods can give >10-fold
variation between two operators. Cells can be different from time to time as
well. Sf9 cells in different labs are quite different. MOI report
Just an offshoot of the same Question..
I would like to ask whether the same applies for GST-tag digestion using
thrombin..
No agitation gives better results in the above case too...
Any personal experiences
On Thu, Mar 31, 2011 at 11:29 AM, Klaus Piontek <
klaus.pion...@ocbc.uni-freiburg.de>
Interesting. My IT, both volume I and volume A (1983) only have P21212 for
space group #18. Do I have to purchase a new volume A every year to keep
up with the new conventions?
Cheers,
Bernie
On Thu, March 31, 2011 12:57 pm, Ian Tickle wrote:
>> I would like to share my experiencde with a rathe
On 3/31/2011 10:14 AM, Jacob Keller wrote:
>> What do we gain? As Dale pointed out, we are already abusing either occupancy, B-factor or delete the side chain to compensate
for our inability to tell the user that the side chain is disordered. With your proposal, we would fudge both occupancy and
> I would like to share my experiencde with a rather unexpected problem of
> indexing conventions. Perhaps I can save people some
> time
> I have a crystal in the more unusual P21212 space-group (No 18). Its unit
> cell lengths are b>a>c (please note). I systematically
> use XDS for data int
While what you say here is quite true and is useful for us to
remember, your list is quite short. I can add another
3) The systematic error introduced by assuming full occupancy for all sites.
There are, of course, many other factors that we don't account for
that our refinement programs ten
> To comment on Phil:
> I fed the mtz-file from pointless into ctruncate (or maybe it was scala) which
> left the space group string (P2 21 21) but turned the space group number 18
> into
> 3018 - this does screw up autosharp and maybe also other programs which use
> the
> space group number/ sym
On Thursday, March 31, 2011 10:05:22 am Hailiang Zhang wrote:
> Dear Zbyszek:
>
> Thanks a lot for your good summary. It is very interesting but, do you
> think there are some references for more detailed description, especially
> from mathematics point of view about correlating B-factor to the Ga
On Thu, Mar 31, 2011 at 10:14 AM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:
> Also,
> setting occupancy = zero is not fudging but rather respectfully
> declining to comment based on lack of data. I think it is exactly the
> same as omitting residues one cannot see in the density.
No
Hi,
We have observed this virus "going off" within 2 months as well. ...any idea
why this happens?
Chitra
On 31 March 2011 18:07, Nathaniel Clark wrote:
> We do adherent if we have a small volume of low titer virus, but as
> soon as we have a decent titer we will start with a 30 ml suspension
>
We don't do anything special to store the virus, just a dark fridge.
You do need to mix well as the virus can settle.
You might want to try this 'TIPS' approach. Basically you freeze
baculovirus infected cells, and use them as your virus innoculum. I
think I tried it once and it didn't really wo
Dear Anita,
I happen to have a very similar problem today.
Does XDS use the desired setting if you provide it with the correct cell and
space group during the IDXREF step? You can otherwise re-index in CORRECT.
To comment on Phil:
I fed the mtz-file from pointless into ctruncate (or maybe it wa
> What do we gain? As Dale pointed out, we are already abusing either
> occupancy, B-factor or delete the side chain to compensate for our inability
> to tell the user that the side chain is disordered. With your proposal, we
> would fudge both occupancy and B-factor, which in my eyes is even wo
Thank you for the comment. You are correct that there are being backups
made in the newer interruptable fit function (they were't previously in
the uninterruptable fitting). I wasnt thinking about that (*). This may
(especially on windows) slow down things a great deal as we write and
gzip these fi
We do adherent if we have a small volume of low titer virus, but as
soon as we have a decent titer we will start with a 30 ml suspension
flask. Normally I add something like 0.5 ml, which is probably more
then I need. We only use serum free media right now. As I mentioned,
if you need to boost t
Dear Zbyszek:
Thanks a lot for your good summary. It is very interesting but, do you
think there are some references for more detailed description, especially
from mathematics point of view about correlating B-factor to the Gaussian
probability distribution (the B-factor unit of A^2 is my first do
This is a lovely summary, and we should make our students read it. - But
I'm afraid I do not see how it supports the closing statement in the
last paragraph... phx.
On 31/03/2011 17:06, Zbyszek Otwinowski wrote:
The B-factor in crystallography represents the convolution (sum) of two
types of
A related question: how do most people amplify their baculovirus
stocks? Adherent cultures vs suspension? Fold dilution at each stage
(P1 to P2, P2 to P3)? Duration of each amplification stage?
We have some viral stocks that "go off" rather quickly (1-2 months)
despite being stored with FBS in a c
Dear Quyen,
On Thu, Mar 31, 2011 at 11:27:58AM -0400, Quyen Hoang wrote:
> Thank you for your post, Herman.
> Since there is no holy bible to provide guidance, perhaps we should hold
> off the idea of electing a "powerful dictator" to enforce this?
> - at least until we all can come to a consensu
Regarding suggestions that the pdb or the IUCR to tell us what to do:
IMO -
Neither of the usual solutions - (a) deleting side chains when there is no
density or (b) letting B factors go where they will - is without problems (this
is clear from the ongoing discussion). I would be really unhapp
The B-factor in crystallography represents the convolution (sum) of two
types of uncertainties about the atom (electron cloud) position:
1) dispersion of atom positions in crystal lattice
2) uncertainty of the experimenter's knowledge about the atom position.
In general, uncertainty needs not to
On Thu, 2011-03-31 at 11:27 -0400, Quyen Hoang wrote:
> Thank you for your post, Herman.
> Since there is no holy bible to provide guidance, perhaps we should
> hold off the idea of electing a "powerful dictator" to enforce this?
> - at least until we all can come to a consensus on how the "dictato
On Thu, 2011-03-31 at 17:04 +0200, herman.schreu...@sanofi-aventis.com
wrote:
> Maybe we should really start using cif files, which allow to specify
> coordinate uncertainties.
PDB has SIGATM record for that purpose
--
"I'd jump in myself, if I weren't so good at whistling."
Thank you for your post, Herman.
Since there is no holy bible to provide guidance, perhaps we should
hold off the idea of electing a "powerful dictator" to enforce this?
- at least until we all can come to a consensus on how the "dictator"
should dictate...
Cheers,
Quyen
On Mar 31, 2011, a
And not at full moon!
Klaus
Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu:
Our experience is do not shake the tube during TEV cleavage,I dont
know why, but it does help.
xiaopeng
Dr. Klaus Piontek
Albert-Ludwigs-University Freiburg
Institute of Organic Chemistry and Biochemistry, Room 401 H
Hi Tom,
You can try the MEROPS database. There is a search feature called "What
peptidases can cleave this bond".
http://merops.sanger.ac.uk/cgi-bin/specsearch.pl
Cheers,
Jack
On Thu, Mar 31, 2011 at 6:59 AM, Brett, Thomas wrote:
> Hi all:
> I was wondering if anyone had any tips on identify
We have the same experience with the DO NOT AGITATE. We purify our own
His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at
about 1 mg/mL.
Laurie Betts
UNC
On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston <
charles.allers...@sgc.ox.ac.uk> wrote:
> I echo this.
>
> So
Dear Jacob,
What do we gain? As Dale pointed out, we are already abusing either occupancy,
B-factor or delete the side chain to compensate for our inability to tell the
user that the side chain is disordered. With your proposal, we would fudge both
occupancy and B-factor, which in my eyes is ev
Well, I guess I was thinking to make the b-factor such a preposterous
value that no one would possibly believe it. Setting occupancies to
zero effectively places a stumbling block, because people see the
residues and think they are actually supported by data. So to
counter-balance this, I thought p
>> - they all know what B is and how to look for regions of high B
>> (with, say, pymol) and they know not to make firm conclusions about H-bonds
>> to flaming red side chains.
>
>But this "knowledge" may be quite wrong. If the flaming red really indicates
>large vibrational motion then yes, on
On Thu, Mar 31, 2011 at 7:42 AM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:
> Why not have the "b-factors take care of it" until some magic cutoff
> number? When they reach the cutoff, two things happen:
>
> 1. Occupancies are set to zero for those side chains, to represent our
> lack o
The IUCr standard is to make ahttp://nvl.nist.gov/pub/nistpubs/jres/107/4/j74mig.pdf
http://nvl.nist.gov/pub/nistpubs/jres/106/6/j66mig.pdf
and P 2 21 21 is a perfectly valid space group
Pointless will reindex within the same point group, or you can choose the a Dear all,
>
> I would like to sha
Why not have the "b-factors take care of it" until some magic cutoff
number? When they reach the cutoff, two things happen:
1. Occupancies are set to zero for those side chains, to represent our
lack of ability to model the region,
2. B-factors are set to exactly 500, as a "flag" allowing casual
I echo this.
Some time ago I was working on a target which seemed to precipitate when
cleaving overnight with TEV. I wasted a fair bit of time trying to optimise
cleavage conditions with a myriad of buffers. In the end, just by not
agitating my solution, there was no precipitation and recove
Dear Javier,
Thanks for your reply. I am using coot 0.6.1, and I could copy molecules
with names besides "A" only if chains are already present in both NCS.
For chains which are only present in one NCS , I did find out a way to copy
to the other NCS by using 'Find NCS ligand' , although it is not
If you are using CCP4, it can accomodate P22121. However, just reindex in
CCP4 to the correct setting with P21212.
Bernie Santarsiero
On Thu, March 31, 2011 9:28 am, Anita Lewit-Bentley wrote:
> Dear all,
>
> I would like to share my experiencde with a rather unexpected problem
> of indexing con
Dear all,
I would like to share my experiencde with a rather unexpected problem
of indexing conventions. Perhaps I can save people some time
I have a crystal in the more unusual P21212 space-group (No 18). Its
unit cell lengths are b>a>c (please note). I systematically use XDS
for dat
Though I'm not meaning to turn this into a plaque assay burn session, for
many reasons we have also abandoned it and instead titer our baculovirus
stocks (P3 and P4) using flow cytometry. We use an antibody against viral
gp64 that has been labeled with PE and stain infected cells in 96 well
plates
Our experience is do not shake the tube during TEV cleavage,I dont know why,
but it does help.
xiaopeng
Dear Quyen,
I am afraid you won't get any better answers than you got so far. There is no
holy bible telling you what to do with disordered side chains. I fully agree
with James that you should try to get the best possible model, which best
explains your data and that will be your decision. Here
Hi all:
I was wondering if anyone had any tips on identifying proteases. I have a
protein for which I know the proteolytic cleavage site. What are the best ways
to identify the protease that does the cutting either:
1) bioinformatically (i.e., a good database to search using the cleavage site
or
Hello all:
I am curious about what level of recovery is reasonable when performing
a TEV cleavage to remove 6HIS tags (N-terminal) from a protein.
Currently we are experiencing >50% loss of soluble protein after TEV
cleavage, and feel that this is too much. Are there better systems for
his tag cle
We are getting off topic a little bit.
Original topic: is it better to not build disordered sidechains or
build them and let B-factors take care of it?
Ed's poll got almost a 50:50 split.
Question still unanswered.
Second topic introduced by Pavel: "Your B-factors are valid within a
harmoni
Judging from some examples in the literature, if there's no density for the
ligand, you publish in Nature!
On 31 Mar 2011, at 14:32, Huanwang Yang wrote:
> If a part of sequence has no density, this part will be cut from coordinates.
> If the side chain of a residue is lack of density, the opin
I checked with someone in our protein production group and got the following
response:
We also stopped doing the virus titration with the plaque assay and instead are
performing expression test with different concentration of virus from the 3rd
amplification. But for some viruses we still have
If a part of sequence has no density, this part will be cut from
coordinates.
If the side chain of a residue is lack of density, the opinion is not
converged.
What about the ligand, if no density is observed?
Huanwang
Ed Pozharski wrote:
The results of the online survey on what to do with di
Dear Zhang yu,
I also found that same problem recently. I think is a small bug from Coot.
It will only copy NCS if the molecule's name you want to copy is molecule A.
So maybe will work if you rename your built chain as A.
Good luck!
Javier
On 31/03/11 00:16, zhang yu wrote:
Dear all,
I have
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