Re: [ccp4bb] crystals that dont diffract :( :(

[Diana Tomchick]

May I add the comment that despite the fact that it would be great if all of 
the samples that I worked with would be stable at room temperature for 3 weeks, 
if I had applied that as a criterion for crystallization screening and 
subsequent structure determination, then my publication record would be about 
1/5 the current length. Most of the really interesting and high-impact 
structures that I’ve worked on over the last 18 years were not that stable, and 
several were ones that were unstable enough that I couldn’t allow the 
crystallization setups to sit for longer than one week without seeing 
degradation of the diffraction pattern.

When you notice this phenomenon, it focuses the mind to search for the most 
optimum conditions for rapid, quality crystal growth. It also relieves one of 
having to wait weeks to months for the best diffracting crystals. You take what 
you can get, and if 3.5 Å is the best you’ll see but it answers your biological 
questions, you’ll be happy with what you have.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Aug 26, 2018, at 1:39 PM, James Holton 
mailto:jmhol...@slac.stanford.edu>> wrote:


An excellent review on improving diffraction that has not been mentioned yet 
is: https://doi.org/10.1107/S0907444905032130

As for how often it happens?  At my beamline we do see this fairly 
infrequently, but often enough that it no longer surprises me. I suppose that 
brings some comfort to the user, but not a lot.

Something important to do in these cases is take an exposure with 180 deg of 
rotation or more on a single image.  This is because you might have a salt 
crystal and happen to have an orientation where no hkls are on the Ewald 
sphere.  Doing the wide sweep will make sure any salt reflections are observed. 
 If you see a beautiful, symmetric pattern of very bright spots, but none 
anywhere near the beamstop, then you've got salt.  Mind you some salts have 
unit cells as long as 10 A or more, but again the wide sweep gives you an upper 
limit on the unit cell size and therefore the unit cell volume and molecular 
weight.  Disappointing to be sure, but better to know that try to optimize it.  
So, in a way, doing a 180-deg shot and still seeing no spots at all is a good 
sign.  Means you have a protein crystal.

Of course, it is possible your 0,0,1 reflections are there and you just can't 
seem them because of the beamstop.  But making the beamstop smaller is probably 
not going to make you any happier.

As for how a protein crystal can not diffract at all?  Yes, it is difficult to 
imagine how a crystal lattice can even exist if the "atomic displacements" are 
so large as to extinguish even the lowest-order reflections.  Those 
"displacements" need to be at least as big as a unit cell or larger to do that. 
 However, for visible light (5000 A wavelength) the unit cell of a protein 
crystal is not very big at all, and movements of a unit cell or more are still 
not enough to perturb an optical photon enough to make the crystal start to 
turn brown from all the little micro-cracks.  So, it is possible to have no 
order in the x-ray range and optical clarity in the visible.  That said, it is 
still hard to imagine how a crystal could _grow_ this way.  It is probably 
something that happens after the lattice forms.  And that is good news.

How does it get that way?  Yes, sitting on the bench for 3 weeks might be why 
your protein is denaturing in the lattice.  Remember, there is nothing magical 
about a crystal lattice.  Yes, they tend to hold things in place, but if your 
protein falls apart after sitting in a tube on the bench after 3 weeks then it 
is not surprising that it might do the same inside a crystal.  One of my user 
labs actually does this as a pre-crystallization assay: leave it out on the 
bench for a few weeks, and then run a gel filtration column again and see if 
its still good.  If not, then you need to work on stability before setting up 
trays.

So, the short answer is that optimization is key.  It is very rare in 
crystallography to get away with not having to do any optimization at all, and 
in those situations you should really worry because your competitor is also 
having an easy time.  It is when things don't go well that being a 
critically-thinking scientist is an advantage.  And that, I hope, is also good 
news.

Good luck!

-James Holton
MAD Scientist


On 8/14/2018 2:58 AM, Careina Edgooms wrote:
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 

Re: [ccp4bb] crystals that dont diffract :( :(

[James Holton]

An excellent review on improving diffraction that has not been mentioned 
yet is: https://doi.org/10.1107/S0907444905032130


As for how often it happens?  At my beamline we do see this fairly 
infrequently, but often enough that it no longer surprises me. I suppose 
that brings some comfort to the user, but not a lot.


Something important to do in these cases is take an exposure with 180 
deg of rotation or more on a single image.  This is because you might 
have a salt crystal and happen to have an orientation where no hkls are 
on the Ewald sphere.  Doing the wide sweep will make sure any salt 
reflections are observed.  If you see a beautiful, symmetric pattern of 
very bright spots, but none anywhere near the beamstop, then you've got 
salt.  Mind you some salts have unit cells as long as 10 A or more, but 
again the wide sweep gives you an upper limit on the unit cell size and 
therefore the unit cell volume and molecular weight.  Disappointing to 
be sure, but better to know that try to optimize it.  So, in a way, 
doing a 180-deg shot and still seeing no spots at all is a good sign.  
Means you have a protein crystal.


Of course, it is possible your 0,0,1 reflections are there and you just 
can't seem them because of the beamstop.  But making the beamstop 
smaller is probably not going to make you any happier.


As for how a protein crystal can not diffract at all?  Yes, it is 
difficult to imagine how a crystal lattice can even exist if the "atomic 
displacements" are so large as to extinguish even the lowest-order 
reflections.  Those "displacements" need to be at least as big as a unit 
cell or larger to do that.  However, for visible light (5000 A 
wavelength) the unit cell of a protein crystal is not very big at all, 
and movements of a unit cell or more are still not enough to perturb an 
optical photon enough to make the crystal start to turn brown from all 
the little micro-cracks.  So, it is possible to have no order in the 
x-ray range and optical clarity in the visible.  That said, it is still 
hard to imagine how a crystal could _grow_ this way.  It is probably 
something that happens after the lattice forms.  And that is good news.


How does it get that way?  Yes, sitting on the bench for 3 weeks might 
be why your protein is denaturing in the lattice.  Remember, there is 
nothing magical about a crystal lattice.  Yes, they tend to hold things 
in place, but if your protein falls apart after sitting in a tube on the 
bench after 3 weeks then it is not surprising that it might do the same 
inside a crystal.  One of my user labs actually does this as a 
pre-crystallization assay: leave it out on the bench for a few weeks, 
and then run a gel filtration column again and see if its still good.  
If not, then you need to work on stability before setting up trays.


So, the short answer is that optimization is key.  It is very rare in 
crystallography to get away with not having to do any optimization at 
all, and in those situations you should really worry because your 
competitor is also having an easy time.  It is when things don't go well 
that being a critically-thinking scientist is an advantage.  And that, I 
hope, is also good news.


Good luck!

-James Holton
MAD Scientist


On 8/14/2018 2:58 AM, Careina Edgooms wrote:
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 harvesting and in liquid nitrogen for about a 
month before diffracting. Could that be a factor? If I regrew more 
beautiful crystals and diffracted straight away could that help?

Careina



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Re: [ccp4bb] crystals that dont diffract :( :(

[Patrick Shaw Stewart]

Hi Careina

Expanding on what Tim says, try crushing your crystals to make a seed
stock, and adding it to a few *random screens - *preferably screens that
you have already tried with this target.

Search for instructions for MMS or rMMS online.

Good luck,

Patrick



On 14 August 2018 at 11:34, Tim Gruene  wrote:

> 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 08/14/2018 11:58 AM, Careina Edgooms wrote:
> > 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 harvesting and in liquid nitrogen for about a month
> > before diffracting. Could that be a factor? If I regrew more beautiful
> > crystals and diffracted straight away could that help?
> > Careina
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
> --
> --
> Paul Scherrer Institut
> Tim Gruene
> - persoenlich -
> OSUA/204
> CH-5232 Villigen PSI
> phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
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[ccp4bb] AW: Re: [ccp4bb] crystals that dont diffract :( :(

[Hughes, Jon]

Yes indeed!
Jon

--
Jon Hughes
(+49/0)1757929098
Sent without the use of Apple products.

 Daniel M. Himmel, Ph. D. schrieb 

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
great deal of intrinsic disorder.  Jon's suggestion to flash-cool them in
oil could work.  Your high PEG concentration in the flash-cooling solution
might work, too, but you probably cannot just plunge your crystals
suddenly into a much higher PEG solution.  A few suggestions:

1) Whichever cryoprotectant you use, introduce it to your crystal GRADUALLY,
such as in steps (e.g., 10% ==> 15% ==> 20% ==>25%) or something like that.
For some proteins, drastic rapid changes of concentrations of anything in
the solution can damage the crystal and introduce enough disorder so that
the protein crystal will not diffract well.  Sometimes you can tell when there's
damage if the crystal cracks (or melts away), but not always.

2) You may have to experiment with different cryoprotectants.  Different
cryoprotectants make different protein crystals happy.  For example, try PEG 
200,
PEG 400, PEG 600, glycerol, sucrose, trehalose, other disaccharide sugars, MPD, 
butanediols.

3) I have found that, as a "rule of thumb", 25% of any cryoprotectant is enough
to protect against ice formation.  If your protein crystal can tolerate it, 
higher
concentrations could be better (because they can shrink the unit cell and reduce
some of the water content).  HOWEVER, many protein crystals will not tolerate
much higher concentrations without taking on damage.

I hope this helps.

-Daniel

On Tue, Aug 14, 2018 at 9:45 AM, ferrer 
mailto:jean-luc.fer...@ibs.fr>> 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 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 harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


--

Jean-Luc Ferrer
Institut de Biologie Structurale
71 Avenue des 
Martyrs
CS 10090
38044 Grenoble Cedex 9 - FRANCE

Ph.:  +33 (0)4 57 42 85 22
Cell: +33 (0)6 89 45 13 57
email: jean-luc.fer...@ibs.fr





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Re: [ccp4bb] crystals that dont diffract :( :(

[Daniel M. Himmel, Ph. D.]

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
great deal of intrinsic disorder.  Jon's suggestion to flash-cool them in
oil could work.  Your high PEG concentration in the flash-cooling solution
might work, too, but you probably cannot just plunge your crystals
suddenly into a much higher PEG solution.  A few suggestions:

1) Whichever cryoprotectant you use, introduce it to your crystal GRADUALLY,
such as in steps (e.g., 10% ==> 15% ==> 20% ==>25%) or something like that.
For some proteins, drastic rapid changes of concentrations of anything in
the solution can damage the crystal and introduce enough disorder so that
the protein crystal will not diffract well.  Sometimes you can tell when
there's
damage if the crystal cracks (or melts away), but not always.

2) You may have to experiment with different cryoprotectants.  Different
cryoprotectants make different protein crystals happy.  For example, try
PEG 200,
PEG 400, PEG 600, glycerol, sucrose, trehalose, other disaccharide sugars,
MPD, butanediols.

3) I have found that, as a "rule of thumb", 25% of any cryoprotectant is
enough
to protect against ice formation.  If your protein crystal can tolerate it,
higher
concentrations could be better (because they can shrink the unit cell and
reduce
some of the water content).  HOWEVER, many protein crystals will not
tolerate
much higher concentrations without taking on damage.

I hope this helps.

-Daniel

On Tue, Aug 14, 2018 at 9:45 AM, ferrer  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 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
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> 
> Jean-Luc Ferrer
> Institut de Biologie Structurale71 Avenue des Martyrs 
> 
> CS 10090
> 38044 Grenoble Cedex 9 - FRANCE
>
> Ph.:  +33 (0)4 57 42 85 22
> Cell: +33 (0)6 89 45 13 57
> email: jean-luc.fer...@ibs.fr
> 
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] crystals that dont diffract :( :(

[ferrer]

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 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 harvesting and in liquid nitrogen for about a 
month before diffracting. Could that be a factor? If I regrew more 
beautiful crystals and diffracted straight away could that help?

Careina



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



--

Jean-Luc Ferrer
Institut de Biologie Structurale
71 Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9 - FRANCE

Ph.:  +33 (0)4 57 42 85 22
Cell: +33 (0)6 89 45 13 57
email: jean-luc.fer...@ibs.fr





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Re: [ccp4bb] crystals that dont diffract :( :(

[zaigham mahmood khan]

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 glitch, and
there was no X-ray. True story!

Best wishes

-Z


Zaigham Mahmood Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York

On Tue, Aug 14, 2018 at 5:58 AM, Careina Edgooms <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> 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
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] crystals that dont diffract :( :(

[Rangana Warshamanage]

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, Andreas Forster 
wrote:

> 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.
> https://www.mitegen.com/product/micrort-room-temperature-starter-kits/
>
> All best.
>
>
> Andreas
>
>
>
> On Tue, Aug 14, 2018 at 12:48 PM, Harry Powell <193323b1e616-dmarc-
> requ...@jiscmail.ac.uk> wrote:
>
>> 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.
>>
>> There are those of us who would contend that if your sample doesn't
>> diffract, then it isn't a crystal, no matter how nice it looks (though it
>> might have been one once...)!
>>
>> Harry
>> --
>> Dr Harry Powell
>> Chairman of European Crystallographic Association SIG9 (Crystallographic
>> Computing)
>>
>>
>>
>>
>> On 14 Aug 2018, at 11:14, Elspeth Garman wrote:
>>
>> 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 [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
>> Of *Hughes, Jon
>> *Sent:* 14 August 2018 11:11
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>>
>> 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 offer this possibility these days.
>> best
>> jon
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
>> ] *Im Auftrag von *Careina Edgooms
>> *Gesendet:* Dienstag, 14. August 2018 11:59
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [ccp4bb] crystals that dont diffract :( :(
>>
>> 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 harvesting and in liquid nitrogen for about a month before
>> diffracting. Could that be a factor? If I regrew more beautiful crystals
>> and diffracted straight away could that help?
>> Careina
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
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>>
>
>
> --
>
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Re: [ccp4bb] crystals that dont diffract :( :(

[Andreas Forster]

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.
https://www.mitegen.com/product/micrort-room-temperature-starter-kits/

All best.


Andreas



On Tue, Aug 14, 2018 at 12:48 PM, Harry Powell <
193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:

> 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.
>
> There are those of us who would contend that if your sample doesn't
> diffract, then it isn't a crystal, no matter how nice it looks (though it
> might have been one once...)!
>
> Harry
> --
> Dr Harry Powell
> Chairman of European Crystallographic Association SIG9 (Crystallographic
> Computing)
>
>
>
>
> On 14 Aug 2018, at 11:14, Elspeth Garman wrote:
>
> 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 [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Hughes,
> Jon
> *Sent:* 14 August 2018 11:11
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>
> 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 offer this possibility these days.
> best
> jon
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
> ] *Im Auftrag von *Careina Edgooms
> *Gesendet:* Dienstag, 14. August 2018 11:59
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [ccp4bb] crystals that dont diffract :( :(
>
> 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
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
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Re: [ccp4bb] crystals that dont diffract :( :(

[Tom Peat]

?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 Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board  on behalf of Careina Edgooms 
<02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Sent: Tuesday, August 14, 2018 7:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystals that dont diffract :( :(

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 harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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Re: [ccp4bb] crystals that dont diffract :( :(

[Harry Powell]

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.

There are those of us who would contend that if your sample doesn't diffract, 
then it isn't a crystal, no matter how nice it looks (though it might have been 
one once...)!

Harry
--
Dr Harry Powell
Chairman of European Crystallographic Association SIG9 (Crystallographic 
Computing) 




On 14 Aug 2018, at 11:14, Elspeth Garman wrote:

> 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 [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
> Jon
> Sent: 14 August 2018 11:11
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>  
> 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 offer this 
> possibility these days.
> best
> jon
>  
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Careina Edgooms
> Gesendet: Dienstag, 14. August 2018 11:59
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [ccp4bb] crystals that dont diffract :( :(
>  
> 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 harvesting 
> and in liquid nitrogen for about a month before diffracting. Could that be a 
> factor? If I regrew more beautiful crystals and diffracted straight away 
> could that help?
> Careina
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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> 
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> 




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Re: [ccp4bb] crystals that dont diffract :( :(

[Tim Gruene]

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 08/14/2018 11:58 AM, Careina Edgooms wrote:
> 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 harvesting and in liquid nitrogen for about a month
> before diffracting. Could that be a factor? If I regrew more beautiful
> crystals and diffracted straight away could that help?
> Careina
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

-- 
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OSUA/204
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A




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Re: [ccp4bb] crystals that dont diffract :( :(

[Elspeth Garman]

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 [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: 14 August 2018 11:11
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(

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 offer this possibility 
these days.
best
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina 
Edgooms
Gesendet: Dienstag, 14. August 2018 11:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] crystals that dont diffract :( :(

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 harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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Re: [ccp4bb] crystals that dont diffract :( :(

[Nikolas]

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, <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> 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
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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