Dear Tom,
don't feel too bad about it - everyone can make a mistake.
Some of the replies give crystallographic tips that may be useful to
other beginning and not-so-beginning crystallographers. Although I
agree the attachments to the first mail would perhaps better be
deleted from the record
perhaps a second table in which certain statistics (Rsym, I/sigma,
CC0.5) are given as a function of, say, 10 bins of resolution would be
more useful than the same table twice at different resolution cutoffs.
then editors, reviewers and ultimately readers can decide for
themselves what resolu
could it be a PEG molecule?
Quoting "Read, Jon":
Anyone see anything like this before? The data is 1.7Angstrom data with
good statistics. The picture shows the solid FoFc density contoured at 3
Sigma in light brown and -3 Sigma in purple. The density is odd as it
appears to be bound to a peptid
possible ugly workaround: renumber the pdb file so where ccp4mg thinks
the N and C-termini are is hidden in the image you are making (if
possible)
if you want to make different views you may need to make differently
renumbered pdb files.
(but probably other people have a smarter way)
as an a
before modelling a long side-chain in non-existing or dubious density,
also make sure it is really there in the protein by sequencing your
expression plasmid. Your arginine (for example) may in fact be a
serine or glycine...databases are not 100% accurate and neither is PCR
if it was used i
We once had a more-or-less MBP-sized fragment before cleavage, but
this turned to be a spontaneous mutation. This expression experiment
had been started from a glycerol stock with an unknown number of
growth cycles prior to expression.
Starting from a fresh transformation with the purified an
apart from optimising the crystallisation conditions, it might be
worth optimising the protein preparation or do some limited
proteolysis - or even express short N-terminal and/or C-terminal
deletions.
Mark
Quoting Patrick Shaw Stewart:
Jahan
It sounds as though the protein crystallizes
...I meant visualisation software of course...
Quoting "VAN RAAIJ , MARK JOHAN":
if this is the first (or second, or third) time you do a DALI
search, take the list output from DALI, start from the top and
superpose each structure with yours and look at the superpositions
if this is the first (or second, or third) time you do a DALI search,
take the list output from DALI, start from the top and superpose each
structure with yours and look at the superpositions with your
favourite superposition software.
This is very educational and the only way you get a feeli
so perhaps the problem indeed is sending different wavelengths as one
file...in our case there is only one crystal, one wavelength, i.e. one
loop, while I clearly submitted all three wavelengths.
Quoting "Miller, Mitchell D.":
We (JCSG) too have been depositing multiple data sets (includi
ok, I stand corrected, Sigma DOES sell it, but under a slightly different name:
81321 FLUKA
Poly(ethylene glycol) methyl ether average Mw 2,000
81321-250G, 23.60 euros
81321-1KG, 71.80 euros
(prices given for Spain)
thanks!
Quoting "VAN RAAIJ , MARK JOHAN":
>
>
&g
Dear All,
is PEG MME 2000 still available in powder form? I think Fluka used to sell it,
but Fluka is no more and Sigma-Aldrich don't sell it.
Hampton Research and Molecular Dimensions (and perhaps others) do sell 50%
(w/v) solutions.
Mark
Mark J van Raaij
Laboratorio M-4
Dpto de Estruc
apart from radation damage it could be a combination of:
- too tight restraints on the B-factors
- 9 sigma not being that much on a the e/A3 scale, i.e. your difference map is
very flat (which is good) and the few peaks that remain stand out a lot, even
if their absolute height is low...
Quot
and using "dice" for the singular "die"
Quoting Robert Blessing:
How can "spectrum" and "spectra" have been overlooked in this thread?
Síocháin! Sláinte!
In Irish: Peace! Health!
Pronounced roughly "Shee'-kahn", "Slawn'-tche".
Bob
Robert H. Blessing, Ph.D.
Senior Research Scientist
another singular/plural grump:
Recently we can read: "phage are".
Phage is singular, the plural is phages (and this does not have that
much to do with latin or greek).
more reading:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109450/
Quoting Paul Emsley:
The PDBe page for 3k78 says:
"The e
typo correction, you'll want the long axis parallel to the rotation
axis, not to the beam.
Mark
Quoting Frank von Delft:
You probably have to tilt your crystal, so that the long axis is
parallel to the beam. We do this routinely: cut a plastic pipette
tip to have a sharp point, then push
Dear Sreetama,
First of all, there are no hard-and-fast "rules" for successful
crystallisation, try changing as many different variables as possible
and go with what works.
Having said that, yes, next I would go for a grid optimisation varying
the pH in 0.2 or 0.5 units over as wide a range
(oops, previous mail got sent before I wanted)
the turtles would be really nice to extend things into 3D.
great find,
Mark
Quoting Phoebe Rice:
Hi all,
For those who teach xtallography - we found some plastic turtles
that can be snapped together in an amazing variety of space groups.
Wo
reminds me of these symmetric 2D P3 lizards:
http://www.worldofescher.com/store/Z51.html
I bought the RGB-coloured set/puzzle after visiting an Escher
exhibition and sometimes use them in crystallography/symmetry teaching.
Nice to make the students assemble them and then decide on the
symmetry
Dear Kavya,
I don't think it is likely you have five MSE and one MET. Rather, I would guess
the side-chain has some disorder, i.e. one or more alternative conformations.
If you don't see density for alternative conformations, the best way to model
the disorder might be partial occupancy of the
agree, any crystallisation idea is worth pursuing, given you have or can make
enough sample to try it with.
having said that, wouldn't you tend to select for the same crystals as the
seed, i.e. crystals of the component on its own?
have you tried limited proteolysis of your sample, incl. a bit of
agree, any crystallisation idea is worth pursuing, given you have or can make
enough sample to try it with.
having said that, wouldn't you tend to select for the same crystals as the
seed, i.e. crystals of the component on its own?
have you tried limited proteolysis of your sample, incl. a bit
did you try looking if there is a company offering data recovery services from
these kind of tapes?
if there is, there may not be a need to buy a tape drive yourself.Mark
Quoting "Min, Xiaoshan":
> Dear CCP4 community,
>
> We have a few datasets that reside in tapes (Sony QG-112M 8 mm tape
>
Dear Armando,
don't know about NACCESS, but I guess it is superseded by AREAIMOL in CCP4
(also in CCP4i); it outputs the accessible volume per atom in the pdb file and
per residue and per chain and some other statistics in the log-file.
Mark
Quoting Armando Albert:
> Does anyone has got some
you can do amino acid analysis on your pure protein, using a commercial or
academic service - I hope these are still around. You should only need to do
this once, then relate the result to your A220, BCA and Bradford assays.
Mark
Quoting Arpit Mishra:
> hello everybody
>
> i am working on the
if the PCR machine is just to be used for standard sub-cloning (amplifying
fragments from other plasmids, cloned cDNA etc.), I would go for the cheapest
one I could find. I guess there are few crystallography projects were the first
PCR step turned out to be the most difficult.
For more sophis
yet, apart from (and additionally to) modelling two conformations of the
side-chain, the B-factor is the only tool we have (now).
Quoting Pavel Afonine:
> Hi Quyen,
>
>
> (...) And if B-factor is an estimate of thermo-motion (or static disorder),
>> then would it not be reasonable to accept th
I observed 3-5 peaks for a baculovirus expressed protein once, and could only
get good crystals if the peaks were crystallised separately, see:
Virology 262, 333-343 (1999)
Virology 262, 333–343 (1999)
Mark
Quoting Ulli Hain:
> Thanks for the suggestions/ideas. The protein is recombinantl
Dear Chen,
It looks like you have a unit cell with two relatively short axes and one long
one - and some disorder. I have experienced a few examples of this with virus
fibre proteins (adenovirus fibre and T4 short tail fibre). For the short tail
fibre we also obtained images in which some line
Hi Sebastiano,
I don't see how the k-off would influence this, given the timescale of growing
crystals.
An explanation in terms of high Kd and relative lack of crystal contacts for
the component with higher temperature factors would sound more convincing to
me.
Mark
Quoting Vellieux Freder
Regarding editorial decisions, I actually welcome editors making more rejection
decisions, i.e. reading the paper before sending it to referees, so I waste
less time, waiting as author, or as referee reading and commenting on papers
for which it would have been clear beforehand that they are no
perhaps we should campaign for it to be obligatory to provide the pdb and
structure factor file to the journal, and thus referees, upon submission? Then
he can look for himself to see that building and refinement have been performed
satisfactorily.
Mark
> Surely the "best" model is the one tha
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