Dear JL,
Years ago, this was a common problem when I was crystallizing myosin
constructs for my doctoral work. Some of the most beautiful crystals I
got showed little or no diffraction. Often this occurs when there is a very
large water content in the asymmetric unit and in proteins that have a
Dear Thomas,
Alternatively you can try shooting on crystals in the drop, in situ. So
fishing, no cryo. But potentially high radiation damage. Can be
considered if you have enough crystals, and if your crystallization
plate makes it possible.
Regards
JL
On 14/08/2018 20:58, Thomas Krey
Hi Amala,
1. Did you verify the sequence or the presence of the His-tag? If you were
not the person for cloning. I used to spend 14 months working on an clone
and eventually I was allowed to check the sequence and verify the
expression of His-tag by Western-Blot. There was no his-tag.
2. You can
I just give an example of oil. Indeed, paraffin is also a very good option.
In some cases (<=5% alcohol). Here is the difference I observed between
paraffin and paratone oil.
1. Paraffin oil has low viscosity. Paratone oil is too sticky with high
viscosity. Sometimes, I mix them with different
I second (third?) what Tommi and Kevin said about using an oil to cover the
drop to slow evaporation (I like paraffin for this—not too viscous). Here’s an
additional nuance: Saturate the oil with the alcohol first, before using it to
cover the drop.
> On 14 Aug 2018, at 2:58 PM, Thomas Krey
Hi Thomas,
This is usual when high volatile solvent is used in crystallization
(membrane or glycoproteins). The crystal may looks very nice with sharp
edges. When you open the cover glass, you may see a very thin film formed
on the hang-on drops. Once you touch the drop, then crystals move very
Hi,
you could try picking in the cold room. Provided the temperature change does
not kill the crystals, this sometimes worked fine for me in similar cases.
Petri
Petri Kursula
--
Professor
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula
Hi, One way is to cover the drop with e.g. paraffin oil (to prevent
evaporation) and fish it out in the oil. Have had luck with that in some cases.
Tommi
On 14 Aug 2018, at 22:09, Bonsor, Daniel
mailto:dbon...@som.umaryland.edu>> wrote:
I have had some luck with adding ~5-10ul of 60%-70%
I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir
to the drop. The larger volume of the drop appears to slow down the whizzing of
the crystals and allows you to get a few crystals. Though it still occurs. You
could also cool the area down or move into the cold room
Dear crystallization experts,
We have 3D protein crystals grown from a microseed matrix screening vapor
diffusion experiment in either
15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2
or in
27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol
Upon opening the corresponding
Dear Amala
Please increase NaCl concentration to 200mM from 50mM. That can help you
out by increasing affinity of your protein to bead and will delay the
elution time.
On Mon, Aug 13, 2018, 7:20 PM amala mathimaran wrote:
> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa
On 14/08/2018 12:27, Careina Edgooms wrote:
Cryoprotectant was 50% PEG just added to the buffer that it
crystallised in. It crystallised under batch.
which kind of batch plate ? Some are not suitable to in situ.
JL
On Tuesday, August 14, 2018, 12:11:17 PM GMT+2, Hughes, Jon
wrote:
Hi
Did you try them at room temp, in situ (straight in the plate). We
observe that time to time on our beamline, when just harvesting, not
mentioning cryo protection, is enough to loose all diffraction. It-s
rare but happens.
Regards
JL
On 14/08/2018 11:58, Careina Edgooms wrote:
I got
Hello Amala,
Usually Ni-NTA won't have this kind of problem of binding. Probably your
protein is have interaction with hexagon his tag which is affecting its
affinity towards beads. You can try putting tag in C- terminal end of the
protein.
On Mon, 13 Aug 2018, 8:02 pm Artem Evdokimov,
wrote:
It does happen that “beautiful” crystals have no diffraction. Here is an
example :
https://www.sciencedirect.com/science/article/pii/0022283691906078
In my experience, crystals are fairly stable. But not always: crystals of human
endothelial nitric oxide synthase or inducible nitric oxide
Hey Careina
I had once experience the same phenomenon. No diffraction at all. That did
not make any sense to me. So i dipped the "empty" loop in the water, and
flash froze the loop in liquid nitrogen. Once exposed to X-ray, there was
no ice rings... Eventually, it turned out that system had a
Hi Careina,
Some other things to check for:
Where did you measure the crystal, on a home source or at a synchrotron? if
at a synchrotron, try to put bit more flux and see whether you see any
diffraction?
Also check the beam and your crystal alignment.
Rangana
On Tue, Aug 14, 2018 at 12:15 PM,
Hi Careina,
if you don't have grey hair (available in the lab), you can still mount
crystals at room temperature. With a MiTeGen RT kit, very little skill is
required to test crystal diffraction or even collect entire data sets at
room temperature.
?We have some crystals that diffract well when fresh (less than one week old)
but lose almost all diffraction by the end of 2 weeks, so age can matter. One
crystals are in liquid nitrogen, they should be safe from further degradation,
but may suffer from ice contamination.
cheers, tom
Tom
Hi
One other thing to try that someone with grey hair in your lab might know about
- mount a "crystal" in a capillary and see if it diffracts at room temp. As
long as you have a source and detector in your home lab, there's no need to go
to a synchrotron and use their in situ facilities.
Dear Careina,
Unfortunately there is no direct connection between nice looking crystals and
good diffraction.
Sometimes ugly crystals can diffract well while nice ones don’t.
You may try crystal dehydration ; it allows to remove excess of solvent and
change crystal packing which can
Dear Careina,
you could use the old crystals, that did not diffract, for microseeding
to regrew nicer crystals. Once you have them, try to use them as quickly
as possible. Three weeks can be a long time for crystals.
Storage in liquid nitrogen should not be the problem.
Best,
Tim
On
Cryoprotectant was 50% PEG just added to the buffer that it crystallised in.
It crystallised under batch.
On Tuesday, August 14, 2018, 12:11:17 PM GMT+2, Hughes, Jon
wrote:
#yiv2688348338 #yiv2688348338 -- _filtered #yiv2688348338
{font-family:Helvetica;panose-1:2 11 6 4 2 2 2 2 2
Yes, essential to test at room temperature without changing their buffer medium
before getting worried!
If they don’t diffract at RT, they are very very unlikely to diffract at
cryotemperatures, whatever you do to them before hand!
Best wishes
Elspeth
From: CCP4 bulletin board
maybe it's the cryobuffer that's the problem (you didn't mention it). you could
try to fish the crystals with minimal liquid attached by mounting them in oil
rather than a cryobuffer. or you could test the native diffraction "in situ"
(at room temperature in the drop): quite a few beamlines
Hello Careina,
Please send pics or didn't happen.
Anyway, in my very short experience, ugly crystals can diffract better than
beautiful ones. But have you checked first if they were protein crystals
or not?
Best of luck,
Nikk
On Tue, 14 Aug 2018, 11:59 Careina Edgooms, <
I got the most beautiful crystals I have ever seen and they don't diffract at
all. Not poor diffraction, NO diffraction. Anyone know why this could be and
how I can go about fixing it? I had three beautiful crystals and not one
diffracted. I did leave them in the drop for about 3 weeks before
Dear All
10 days to the registration closure for the second CCP4/Spring-8 (RIKEN)
workshop running from 1-6 October 2018.
This workshop will cover data collection, processing and structure solution.
Details are available at
http://www.ccp4.ac.uk/schools/Japan-2018 , with more to follow.
Dear all,
I would like to bring to your attention a postdoctoral position in my research
group at NNF-CPR, University of Copenhagen, to study the structure and function
of contractile injection systems targeting eukaryotic cells. The position is
fully funded and is for 2 years in the first
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