Dear CCP4bb,
Slightly off topic but perhaps of interest to students.
Synopsis:
The MARCO (MAchine Recognition of Crystallization Outcomes) project showed that
automated image analysis can be used to classify experimental results, having a
94% correct classification rate. Work on a custom image
Dear CCP4bb,
From my industrial perspective: The crystallisation bottleneck has probably
been fairly well addressed. High throughput screening using robotics and around
20ul of protein per screen is common. There are lots of screens to try even if
they are rather redundant in their content. How
Hi Firdous,
I had success last week with a condition containing 1.5M MgCl2 which I’d tried
cryo-ing with high salt before and got no diffraction.
Adding dimethylsulphoxide to a final concentration of 10% v/v in the well
solution worked well for me. DMSO was bound in the structure which can be a
Maybe this has already been mentioned in which case apologies:
I’d try rapid dilution. My only experience of having to refold worked both by
dialysis and the dilution method but dilution had the advantage of allowing
various conditions to be tested quickly and more controllably at small scale. I
Hi Praveen,
20 years ago I seemed to spend more time siliconising cover slips than setting
up the Magic 50. We had special jigs to hold a couple of dozen slips for
washing, drying and finally immersion in silane solution. This method was very,
very tedious. An alternative method was to pull a v
I have been presented with crystals grown in nmr tubes on two separate
occasions. Both diffracted very well and thankfully, neither required a
magnetic field for optimisation. On the first occasion I did use the initial
sample as a seed stock.
Best wishes,
David
From: CCP4 bulletin board [mai
Hi Weifei,
When I was looking at Holliday junction annealing I found that the salt
concentration was important. NaCl around 300-500mM improved yield as did MgCl2
at lower (100-200mM) concentrations. So, it might be worth trying higher salt.
Dave
-Original Message-
From: CCP4 bulletin b
You could try adding TEV to your protein just prior to crystallisation. I
produced 2 equivalent structures this way; one with the tag clearly visible
packing between molecules in an I centred space group and a 2nd (with TEV
added) in P21 where the tag was no longer visible (the packing suggeste
The crystals don't look that bad in my opinion. Maybe the cryo step is sub
optimal? Try using large loops (reduces surface tension forces during
transfer). Butane 2,3 diol is a good cryo to try. Maybe shoot them at room temp
(in situ) to get an idea of how they diffract before you manipulate the
On the face of it, I wouldn’t get overly excited by the drop. However, I’ve
been underwhelmed and wrong before. If you’ve got access to a synchrotron
beamline that can shoot the material in-situ it might be a good experiment to
try.
Best of luck,
Dave
Dr. David Hargreaves
Associate Principal Sc
Hi Reza,
I can recommend the work of Marc-Olivier Coppens and Kourosh Malek and the
references therein for an interesting journey through solvent channels.
David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery S
Dear CCP4bb,
I'm refining an antibody structure which requires Kabat residue numbering with
insertion codes. My setup of Refmac5 and Buster both break peptide bonds
between some (not all) of the residues with insertion codes. I was wondering
whether there is a special way of handling these resi
Hi All,
I had an interesting case recently where a Cl atom of a chlorophenyl moiety
went missing in a structure (primary X-ray damage was the suspected culprit).
Small molecule Mass Spec suggested the atom was there to start with but it was
quite obviously missing in the maps (1.9Ang) and the d
Dear CCP4bb,
Does anyone know a simple way to get rid of residue number insertion codes and
then renumber residues normally in pdb files?
Help much appreciated,
David
David Hargreaves
Associate Principal Scientist
_
AstraZene
Dear CCP4bb,
Does anyone know who (if anyone) supports Crystal Miner (Emerald / Decode).
Kind Regards,
David
David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure & Biophysics
Mereside, 50F4
Dear CCP4bbers,
I was wondering whether anyone knew of a program that would calculate
the pI of a protein based on its structure rather than its sequence and
whether such a calculation might correlate better with the measured pI
Dave
David Hargreaves
Associate Principal Scientist
_
Dear Careina,
I would be cautious of using dyes. Much better to 1) try in-situ
diffraction if possible as this is least invasive or 2) pick a sensible
cryo and just freeze the crystal(s). I would try to same something from
the experiment for seed stock & possibly sequencing.
Dave
David Har
Judith,
If you can, try monitoring the diffraction during the dehydration experiment.
There are plenty of ways of doing this. Perhaps the simplest is to shoot your
crystals in-situ after swapping the precipitant for a more concentrated one.
The is also the Free Mounting System.
David Hargreaves
Hi Yuri,
I've had a very similar experience last week which ended badly. I opted to cryo
my new crystals using my favourite cryo. Diffraction showed at least the
crystals were protein but not much more. A classic example of cryo killing
crystals. I mounted the remaining tiny fragment from the dr
I've seen this effect too. In the case I saw the drops were growing (and
spreading) due to a reverse diffusion gradient. Optimised conditions used
"salting in" method i.e. crystallising as the [pptnt] decreases during
equilibration due to dilution as water is Tx from well to drop. Rare but not
Hi Zhao,
I would try a crystal that has not been treated with any dye. Small
molecules, particularly drug like compounds, crystallise out in soaking
experiments and can give diffraction similar to yours. My worry is that
you have non-diffracting protein crystals coated in some small molecule
cry
There are plenty of reports (some even successful) where small concentrations
of proteases have been used to enable crystallisation (with soluble proteins).
Having put the effort into getting your material in the first place it's a
small extra step to try. The professional way is to run a time c
In trying to reproduce a very nice public structure a cloning mis-hap put a
-GS- prior to the C-term 6His tag. The resultant crystals had a 500Ang C
dimension and 16 molecules in the au. Even a single amino acid could make all
the difference
David Hargreaves
Associate Principal Scientist
__
I've collected data from crystal soaked with brightly coloured ligands
which partition into the solvent channels. Although the crystal becomes
highly coloured (mopping up most of the colour from the drop) the
structures remained resolutely empty. I am intrigued by this observation
and wonder how mu
I went through the peg solid stocks in our cupboard and noticed some of them
smelled acrid. I probably won’t use them. Not sure whether they’re chemically
decomposing. Maybe fossilising?
Dave
David Hargreaves
Associate Principal Scientist
_
Hi Aidong,
I've seen this in protein samples that have been snap frozen then thawed on
ice. Thawing at room temperature (or in the hand) stopped this state forming.
Could temperature be the issue? Maybe try 10C or leave the samples on the bench
for a while and see if the state is reversible?
D
Hi Andrea,
If you haven't already done so it might be worth trying a room temperature
mount (capillary or in-situ) to get a feeling for how well the crystals
diffract to start with.
Dave
David Hargreaves
Associate Principal Scientist
Dear CCP4,
I'm looking for the authors of the programs (listed below). I want to
analyse our crystal systems and would like to use these algorithms.
However, I'm not convinced the addresses on the papers are current.
Does anybody know how to contact the authors or of any other programs
that exist
Hi Timur,
I've had a crystal which sounds similar to yours. When I came to freeze
it noticed that it was not exactly solid more amorphous like treacle.
Laughingly I went ahead and screened it anyway and was shocked to see
diffraction to around 15Ang. I was lucky enough to have other crystals
in
p you have), followed
by a second call using symop/symcommit to apply unit cell translations.
Cheers
Martyn
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Hargreaves, David
Sent: 30 June 2011 13:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] generate large symmetry mod
Does anyone have a rigorous method (or script) for generating an
extended lattice e.g 3x3x3 unit cells from any pdb file?
Any help gratefully received,
Dave
David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DE
Take two siliconised glass cover slips. Place 2-5ul of mother liquor on one and
transfer a single crystal to this solution. Place the other cover slip on top
and press down. The two optically flat surfaces crush the crystal (best
observed using a microscope). Slide the two cover slips apart and
Dear CCP4bb,
I like to take this opportunity to draw your attention to the following role
(below).
Please follow the link to the AstraZeneca web site (job search DECS156) for
more details and application forms.
(please don't apply via this email!)
David Hargreaves
##
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