Re: [ccp4bb] Fragile Crystals

[Nicolas Foos]

Dear Morgan Elizabeth,


To complete the answers and suggestion made by the ccp4 community, I 
would add, that you can actually combine the service offered by HTX 
facility : You send your protein, and they are preparing the 
crystallization plate for you in CD-plate which may be use in both 
Harvester and for Direct in-situ data collection (no harvesting, nothing 
except the X-ray will touch the crystals). The great advantage is that 
like this, you don't have to be worried by the plate transportation. 
They are prepared on-site and then if you go for an in-situ experiment, 
it can be measured on three of the ESRF beamlines. Notably, we can on 
MASSIF-1 combine in-situ screening then if required automated harvesting 
directly at the beamline.



Feel free to contact us.

mbow...@embl.fr

didier.nuri...@esrf.fr

marq...@embl.fr


All the best,

Nicolas




On 23/11/2023 10:31, Jose A. MARQUEZ wrote:


Dear Elizabeth,


/In situ/ data collection is a good approach to try in your case. You 
could use the CrystalDirect technology for automated crystal 
harvesting that is more gentle to crystals than manual harvesting. 
This is  available both at EMBL Grenoble and EMBL Hamburg facilities, 
which offer integrated crystallography services in collaboration with 
the ESRF and Petra III synchrotrons.



doi:10.3791/62491.

doi:10.1016/j.crmeth.2021.100102.

doi:10.1107/s0907444912031459.


Best wishes


Josan

_
Jose A. Marquez, Senior Scientist
Head of the Crystallization Facility
European Molecular Biology Laboratory, Grenoble.
Delivery address: EMBL, 71, Avenue des Martyrs
38000 Grenoble, France
Postal address: EMBL, 71, Avenue des Martyrs
CS 90181 38042 Grenoble Cedex 9, France
Phone +33 (0)476 20 74 25
Fax. +33 (0)476 20 71 99

https://www.embl.org/groups/marquez/  
https://www.embl.org/services-facilities/grenoble/high-throughput-crystallisation/  
https://htxlab.embl.org/  
_

On 11/22/2023 5:44 PM, Blake, Morgan Elizabeth wrote:

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and 
I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M 
BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow 
to a large size (greater than 0.5 mm), but the crystals seem to be 
very fragile. When we open the drops to harvest the crystals, we have 
little time to harvest the crystals before they crack. When we move 
the crystals to a cryoprotectant, over time they start fracturing. 
We've tried using different percentages of glycerol, ethylene glycol, 
PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization 
conditions, but we seem to get the same morphology in other 
conditions. Does anyone have suggestions for additives we could use 
post-crystallization to help stabilize our crystals?


Thanks for your advice!



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--
Nicolas Foos PhD - ARISE fellow
https://orcid.org/-0003-2331-8399
   
EMBL Grenoble, McCarthy Team

71 av. des Martyrs,
38000 Grenoble FRANCE
   
+33 4 57 42 84 67




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Re: [ccp4bb] Fragile Crystals

[Dom Bellini]

Hi Morgan Elizabeth,

You could try this protocol 
<https://scripts.iucr.org/cgi-bin/paper?S1600576722002382> (paper title 
below, shameless plug), it can cryo-protect your crystals without the 
need of touching the crystal drop, while at the same time it is likely 
to make your crystals more robust and diffracting to higher resolution.



 A drug-discovery-oriented non-invasive protocol for protein
 crystal cryoprotection by dehydration, with application for
 crystallization screening
 <https://scripts.iucr.org/cgi-bin/paper?S1600576722002382>


Good luck!

D




On 23/11/2023 09:52, SHEPARD William wrote:


CAUTION: This email originated from outside of the LMB.
Do not click links or open attachments unless you recognize the sender 
and know the content is safe.

*.-owner-ccp...@jiscmail.ac.uk-.*

Hi Morgan,
Cases like yours are the motivation why the PROXIMA 2A beamline 
(amongst other beamlines) have developed a plate screener for 
collecting X-ray diffraction data in situ. I suggest you contact those 
beamlines to find out how to get your plates in the beam.

Cheers,
Bill


*From: *"Blake, Morgan Elizabeth" 
*To: *"CCP4BB" 
*Sent: *Wednesday, 22 November, 2023 17:44:29
*Subject: *[ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and 
I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M 
BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow 
to a large size (greater than 0.5 mm), but the crystals seem to be 
very fragile. When we open the drops to harvest the crystals, we have 
little time to harvest the crystals before they crack. When we move 
the crystals to a cryoprotectant, over time they start fracturing. 
We've tried using different percentages of glycerol, ethylene glycol, 
PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization 
conditions, but we seem to get the same morphology in other 
conditions. Does anyone have suggestions for additives we could use 
post-crystallization to help stabilize our crystals?


Thanks for your advice!



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






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Re: [ccp4bb] Fragile Crystals

[SHEPARD William]

Hi Morgan, 
Cases like yours are the motivation why the PROXIMA 2A beamline (amongst other 
beamlines) have developed a plate screener for collecting X-ray diffraction 
data in situ. I suggest you contact those beamlines to find out how to get your 
plates in the beam. 
Cheers, 
Bill 


From: "Blake, Morgan Elizabeth"  
To: "CCP4BB"  
Sent: Wednesday, 22 November, 2023 17:44:29 
Subject: [ccp4bb] Fragile Crystals 

Hello! 

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals? 

Thanks for your advice! 




To unsubscribe from the CCP4BB list, click the following link: 
[ https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 | 
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Re: [ccp4bb] Fragile Crystals

[Harry Powell]

Hi

Well worth contacting your favourite (or favorite…) synchrotron about in-plate 
data collection - I’d imagine that APS has something in-place (there was a 
report way back in the depths of time [2015, when I still worked in the field…] 
about a prototype on ID-19?), and that might be easier to get samples to (from 
Birmingham) than one of the European facilities.

Harry

> On 23 Nov 2023, at 09:31, Jose A. MARQUEZ  wrote:
> 
> Dear Elizabeth,
> 
> In situ data collection is a good approach to try in your case. You could use 
> the CrystalDirect technology for automated crystal harvesting that is more 
> gentle to crystals than manual harvesting. This is  available both at EMBL 
> Grenoble and EMBL Hamburg facilities, which offer integrated crystallography 
> services in collaboration with the ESRF and Petra III synchrotrons. 
> 
> doi:10.3791/62491. 
> doi:10.1016/j.crmeth.2021.100102. 
> doi:10.1107/s0907444912031459.
> 
> Best wishes
> 
> Josan
> _ 
> Jose A. Marquez, Senior Scientist 
> Head of the Crystallization Facility 
> European Molecular Biology Laboratory, Grenoble. 
> Delivery address: EMBL, 71, Avenue des Martyrs 
> 38000 Grenoble, France 
> Postal address: EMBL, 71, Avenue des Martyrs 
> CS 90181 38042 Grenoble Cedex 9, France 
> Phone +33 (0)476 20 74 25 
> Fax. +33 (0)476 20 71 99 
> 
> 
> https://www.embl.org/groups/marquez/
>  
> 
> https://www.embl.org/services-facilities/grenoble/high-throughput-crystallisation/
>  
> 
> https://htxlab.embl.org/
>  
> _
> 
> On 11/22/2023 5:44 PM, Blake, Morgan Elizabeth wrote:
>> Hello!
>> 
>> I am a PhD student working on a crystallography project to wrap up my 
>> dissertation research. I have purified a complex of two proteins, and I can 
>> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane 
>> pH 7.5. These crystals have sharp edges and can grow to a large size 
>> (greater than 0.5 mm), but the crystals seem to be very fragile. When we 
>> open the drops to harvest the crystals, we have little time to harvest the 
>> crystals before they crack. When we move the crystals to a cryoprotectant, 
>> over time they start fracturing. We've tried using different percentages of 
>> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no 
>> success. Needless to say, the crystals do not diffract well, with spot 
>> patterns that look very streaky/mosaic, which I presume is due to the 
>> defects that we see in harvesting/handling. We have screened for alternate 
>> crystallization conditions, but we seem to get the same morphology in other 
>> conditions. Does anyone have suggestions for additives we could use 
>> post-crystallization to help stabilize our crystals?
>> 
>> Thanks for your advice!
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Fragile Crystals

[Jose A. MARQUEZ]

Dear Elizabeth,


/In situ/ data collection is a good approach to try in your case. You 
could use the CrystalDirect technology for automated crystal harvesting 
that is more gentle to crystals than manual harvesting. This is  
available both at EMBL Grenoble and EMBL Hamburg facilities, which offer 
integrated crystallography services in collaboration with the ESRF and 
Petra III synchrotrons.



doi:10.3791/62491.

doi:10.1016/j.crmeth.2021.100102.

doi:10.1107/s0907444912031459.


Best wishes


Josan

_
Jose A. Marquez, Senior Scientist
Head of the Crystallization Facility
European Molecular Biology Laboratory, Grenoble.
Delivery address: EMBL, 71, Avenue des Martyrs
38000 Grenoble, France
Postal address: EMBL, 71, Avenue des Martyrs
CS 90181 38042 Grenoble Cedex 9, France
Phone +33 (0)476 20 74 25
Fax. +33 (0)476 20 71 99

https://www.embl.org/groups/marquez/  
https://www.embl.org/services-facilities/grenoble/high-throughput-crystallisation/  
https://htxlab.embl.org/  
_


On 11/22/2023 5:44 PM, Blake, Morgan Elizabeth wrote:

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and 
I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M 
BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow 
to a large size (greater than 0.5 mm), but the crystals seem to be 
very fragile. When we open the drops to harvest the crystals, we have 
little time to harvest the crystals before they crack. When we move 
the crystals to a cryoprotectant, over time they start fracturing. 
We've tried using different percentages of glycerol, ethylene glycol, 
PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization 
conditions, but we seem to get the same morphology in other 
conditions. Does anyone have suggestions for additives we could use 
post-crystallization to help stabilize our crystals?


Thanks for your advice!



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







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Re: [ccp4bb] Fragile Crystals

[Hough, Michael (DLSLtd,RAL,LSCI)]

Hi Elizabeth,

As suggested VMXi at Diamond may be able to help with in situ data collection 
without harvesting. I'd be happy to discuss if useful?

cheers

Mike

Sent from Outlook for Android<https://aka.ms/AAb9ysg>

From: CCP4 bulletin board  on behalf of CCP4BB 
<193323b1e616-dmarc-requ...@jiscmail.ac.uk>
Sent: Thursday, November 23, 2023 7:04:45 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Fragile Crystals

Hi

Sorry if someone else has mentioned this already, but how about shooting your 
crystals in situ (i.e. while sitting in the plate)? I know that VMXi at Diamond 
can do this (in fact it's designed for his purpose) but haven't collected data 
in long enough not to know if it can be done elsewhere.

Harry
--
Dr Harry Powell

On 22 Nov 2023, at 20:04, Phil Jeffrey 
mailto:pjeff...@princeton.edu>> wrote:

Hello Morgan

In addition to the other good suggestions, I have a few observations of my own.

If your crystals crack without handling or adding anything to the drop, then 
they are extremely environment-sensitive.  If that's the case, testing at room 
temperature will be problematic because that tends to be somewhat stressful on 
the crystal either mechanically (ye olde capillary mount method) or via 
dehydration (loop mounts with the sleeve).

Growing in the presence of at least a little cryoprotectant as per Vaheh would 
be less stressful than multi-step processes like Tao-Hsin's advice unless your 
crystals can re-anneal after stress.  Mounting directly from the drop is 
probably essential, and mounting under oil is a good thing to try in addition - 
apart from anything else oil on the drop slows down the environmental changes.  
Using Mitegen mounts might be less stressful on some crystals than standard 
nylon loops if they are mechanically sensitive.  Spending some time optimizing 
the mechanics of your freezing technique might help significantly in reducing 
the amount of time your crystal dehydrates while moving through air.
(Jim Pflugrath's article is full of useful information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )

Small crystals often freeze more smoothly than large ones - even for robust 
crystals like tetragonal lysozyme.  Try a lot of crystals - I've had projects 
were two different crystals in the same loop from the same drop showed 
radically different diffraction.  Also I've encountered several cases where the 
appearance of disorder varies within a crystal when using a microfocus 
synchrotron beam line (I mostly use FMX and AMX at NSLS2).

Lastly, really cranky crystals rings a distant bell of something we encountered 
in the p19(INK4d)-Cdk6 structure back in the 1990's.  I think it was Jie-Oh Lee 
that did the hard work on this, but in many instances crystals cracked in situ 
when merely opening the drop, and the fix was by adding a cross-linker to the 
well, resealing the drop and waiting for the cross-linker to diffuse:

"The crystals were pretreated with glutaraldehyde (diffused into the drop from 
a reservoir of 30% glutaraldehyde) to reduce their tendency to crack and lose 
diffraction along b* and c*."
https://www.nature.com/articles/26155#Sec9

Most crystals don't love being cross-linked, and I would call this a successful 
instance of a desperation maneuver.

Good luck.
Phil Jeffrey
Princeton

On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:
Hello
I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?
Thanks for your advice!

To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
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Re: [ccp4bb] Fragile Crystals

[CCP4BB]

Hi

Sorry if someone else has mentioned this already, but how about shooting your 
crystals in situ (i.e. while sitting in the plate)? I know that VMXi at Diamond 
can do this (in fact it's designed for his purpose) but haven't collected data 
in long enough not to know if it can be done elsewhere.

Harry
--
Dr Harry Powell

> On 22 Nov 2023, at 20:04, Phil Jeffrey  wrote:
> 
> Hello Morgan
> 
> In addition to the other good suggestions, I have a few observations of my 
> own.
> 
> If your crystals crack without handling or adding anything to the drop, then 
> they are extremely environment-sensitive.  If that's the case, testing at 
> room temperature will be problematic because that tends to be somewhat 
> stressful on the crystal either mechanically (ye olde capillary mount method) 
> or via dehydration (loop mounts with the sleeve).
> 
> Growing in the presence of at least a little cryoprotectant as per Vaheh 
> would be less stressful than multi-step processes like Tao-Hsin's advice 
> unless your crystals can re-anneal after stress.  Mounting directly from the 
> drop is probably essential, and mounting under oil is a good thing to try in 
> addition - apart from anything else oil on the drop slows down the 
> environmental changes.  Using Mitegen mounts might be less stressful on some 
> crystals than standard nylon loops if they are mechanically sensitive.  
> Spending some time optimizing the mechanics of your freezing technique might 
> help significantly in reducing the amount of time your crystal dehydrates 
> while moving through air.
> (Jim Pflugrath's article is full of useful information:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )
> 
> Small crystals often freeze more smoothly than large ones - even for robust 
> crystals like tetragonal lysozyme.  Try a lot of crystals - I've had projects 
> were two different crystals in the same loop from the same drop showed 
> radically different diffraction.  Also I've encountered several cases where 
> the appearance of disorder varies within a crystal when using a microfocus 
> synchrotron beam line (I mostly use FMX and AMX at NSLS2).
> 
> Lastly, really cranky crystals rings a distant bell of something we 
> encountered in the p19(INK4d)-Cdk6 structure back in the 1990's.  I think it 
> was Jie-Oh Lee that did the hard work on this, but in many instances crystals 
> cracked in situ when merely opening the drop, and the fix was by adding a 
> cross-linker to the well, resealing the drop and waiting for the cross-linker 
> to diffuse:
> 
> "The crystals were pretreated with glutaraldehyde (diffused into the drop 
> from a reservoir of 30% glutaraldehyde) to reduce their tendency to crack and 
> lose diffraction along b* and c*."
> https://www.nature.com/articles/26155#Sec9
> 
> Most crystals don't love being cross-linked, and I would call this a 
> successful instance of a desperation maneuver.
> 
> Good luck.
> Phil Jeffrey
> Princeton
> 
>> On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:
>> Hello
>> I am a PhD student working on a crystallography project to wrap up my 
>> dissertation research. I have purified a complex of two proteins, and I can 
>> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane 
>> pH 7.5. These crystals have sharp edges and can grow to a large size 
>> (greater than 0.5 mm), but the crystals seem to be very fragile. When we 
>> open the drops to harvest the crystals, we have little time to harvest the 
>> crystals before they crack. When we move the crystals to a cryoprotectant, 
>> over time they start fracturing. We've tried using different percentages of 
>> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no 
>> success. Needless to say, the crystals do not diffract well, with spot 
>> patterns that look very streaky/mosaic, which I presume is due to the 
>> defects that we see in harvesting/handling. We have screened for alternate 
>> crystallization conditions, but we seem to get the same morphology in other 
>> conditions. Does anyone have suggestions for additives we could use 
>> post-crystallization to help stabilize our crystals?
>> Thanks for your advice!
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> 

Re: [ccp4bb] Fragile Crystals

[Phil Jeffrey]

Hello Morgan

In addition to the other good suggestions, I have a few observations of 
my own.


If your crystals crack without handling or adding anything to the drop, 
then they are extremely environment-sensitive.  If that's the case, 
testing at room temperature will be problematic because that tends to be 
somewhat stressful on the crystal either mechanically (ye olde capillary 
mount method) or via dehydration (loop mounts with the sleeve).


Growing in the presence of at least a little cryoprotectant as per Vaheh 
would be less stressful than multi-step processes like Tao-Hsin's advice 
unless your crystals can re-anneal after stress.  Mounting directly from 
the drop is probably essential, and mounting under oil is a good thing 
to try in addition - apart from anything else oil on the drop slows down 
the environmental changes.  Using Mitegen mounts might be less stressful 
on some crystals than standard nylon loops if they are mechanically 
sensitive.  Spending some time optimizing the mechanics of your freezing 
technique might help significantly in reducing the amount of time your 
crystal dehydrates while moving through air.

(Jim Pflugrath's article is full of useful information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ )

Small crystals often freeze more smoothly than large ones - even for 
robust crystals like tetragonal lysozyme.  Try a lot of crystals - I've 
had projects were two different crystals in the same loop from the same 
drop showed radically different diffraction.  Also I've encountered 
several cases where the appearance of disorder varies within a crystal 
when using a microfocus synchrotron beam line (I mostly use FMX and AMX 
at NSLS2).


Lastly, really cranky crystals rings a distant bell of something we 
encountered in the p19(INK4d)-Cdk6 structure back in the 1990's.  I 
think it was Jie-Oh Lee that did the hard work on this, but in many 
instances crystals cracked in situ when merely opening the drop, and the 
fix was by adding a cross-linker to the well, resealing the drop and 
waiting for the cross-linker to diffuse:


"The crystals were pretreated with glutaraldehyde (diffused into the 
drop from a reservoir of 30% glutaraldehyde) to reduce their tendency to 
crack and lose diffraction along b* and c*."

https://www.nature.com/articles/26155#Sec9

Most crystals don't love being cross-linked, and I would call this a 
successful instance of a desperation maneuver.


Good luck.
Phil Jeffrey
Princeton

On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote:

Hello

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I 
can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS 
propane pH 7.5. These crystals have sharp edges and can grow to a large 
size (greater than 0.5 mm), but the crystals seem to be very fragile. 
When we open the drops to harvest the crystals, we have little time to 
harvest the crystals before they crack. When we move the crystals to a 
cryoprotectant, over time they start fracturing. We've tried using 
different percentages of glycerol, ethylene glycol, PEG400, and oil for 
cryoprotectants with no success. Needless to say, the crystals do not 
diffract well, with spot patterns that look very streaky/mosaic, which I 
presume is due to the defects that we see in harvesting/handling. We 
have screened for alternate crystallization conditions, but we seem to 
get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help 
stabilize our crystals?


Thanks for your advice!



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Re: [ccp4bb] Fragile Crystals

[Tao-Hsin Chang]

Hi Morgan Elizabeth,

Just following up on Vaheh’s suggestion, you can also add 2-4% of 
cryoprotectant, which includes your mother liquor (and your protein if crystals 
have dissolved), into the crystallization drop daily or weekly after crystals 
appear. Repeat this step until reaching the desired concentration of 
cryoprotectant. Additionally, I will harvest smaller crystals in your case.

If you can test the diffraction at room temperature, it seems to be the first 
priority for me to determine whether it is worthwhile to spend time on this 
crystallization condition and construct.

Good luck!

Best wishes,
Tao-Hsin

> On Nov 22, 2023, at 12:26 PM, Oganesyan, Vaheh 
>  wrote:
> 
> Hi Morgan Elizabeth,
>  
> In some cases adding 1-2% of cryoprotectant into crystallization drop during 
> setting those drops up helps to introduce 25-30% of the same cryoprotectant 
> during harvest, provided you still can get those crystals to grow. Worked for 
> me in several cases.
>  
> Vaheh
>  
> From: CCP4 bulletin board  <mailto:CCP4BB@JISCMAIL.AC.UK>> On Behalf Of Blake, Morgan Elizabeth
> Sent: Wednesday, November 22, 2023 11:44 AM
> To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> Subject: [ccp4bb] Fragile Crystals
>  
> Hello!
>  
> I am a PhD student working on a crystallography project to wrap up my 
> dissertation research. I have purified a complex of two proteins, and I can 
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane 
> pH 7.5. These crystals have sharp edges and can grow to a large size (greater 
> than 0.5 mm), but the crystals seem to be very fragile. When we open the 
> drops to harvest the crystals, we have little time to harvest the crystals 
> before they crack. When we move the crystals to a cryoprotectant, over time 
> they start fracturing. We've tried using different percentages of glycerol, 
> ethylene glycol, PEG400, and oil for cryoprotectants with no success. 
> Needless to say, the crystals do not diffract well, with spot patterns that 
> look very streaky/mosaic, which I presume is due to the defects that we see 
> in harvesting/handling. We have screened for alternate crystallization 
> conditions, but we seem to get the same morphology in other conditions. Does 
> anyone have suggestions for additives we could use post-crystallization to 
> help stabilize our crystals?
>  
> Thanks for your advice!
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>
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Re: [ccp4bb] Fragile Crystals

[Oganesyan, Vaheh]

Hi Morgan Elizabeth,

In some cases adding 1-2% of cryoprotectant into crystallization drop during 
setting those drops up helps to introduce 25-30% of the same cryoprotectant 
during harvest, provided you still can get those crystals to grow. Worked for 
me in several cases.

Vaheh

From: CCP4 bulletin board  On Behalf Of Blake, Morgan 
Elizabeth
Sent: Wednesday, November 22, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>



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Re: [ccp4bb] Fragile Crystals

[srikannathasan velupillai]

Hi Blake,
 You can cross seed and get new condition with good stable crystal
system. Also you could try VMXi beamline, you do not need to use cryo and
use the plate directly.. You have to use Greiner Crystal QuickX ™ or
MiTeGen In-Situ-1 ™ plates.

Thanks Kannan
Show quoted text

On Wed, 22 Nov 2023, 16:54 Blake, Morgan Elizabeth,  wrote:

> Hello!
>
> I am a PhD student working on a crystallography project to wrap up my
> dissertation research. I have purified a complex of two proteins, and I can
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane
> pH 7.5. These crystals have sharp edges and can grow to a large size
> (greater than 0.5 mm), but the crystals seem to be very fragile. When we
> open the drops to harvest the crystals, we have little time to harvest the
> crystals before they crack. When we move the crystals to a cryoprotectant,
> over time they start fracturing. We've tried using different percentages of
> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no
> success. Needless to say, the crystals do not diffract well, with spot
> patterns that look very streaky/mosaic, which I presume is due to the
> defects that we see in harvesting/handling. We have screened for alternate
> crystallization conditions, but we seem to get the same morphology in other
> conditions. Does anyone have suggestions for additives we could use
> post-crystallization to help stabilize our crystals?
>
> Thanks for your advice!
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Fragile Crystals

[Sarah Bowman]

Hi Morgan,

Some of the things that you could try with the conditions that you have already 
found:

1) Lower the temperature for incubation, which might slow down crystal 
formation and decrease potential defects as the crystals form

2) Harvest the crystals using a humidity control device like the Watershed from 
MiTeGen

3) Try adding oil to the top of the well and harvest through the oil as 
cryoprotectant

4) Try different cryos and different methods of adding cryoprotectant

5) Try collecting at room temperature – that may help identify if the issue is 
the crystals or the cryo

Finally, you may need to find other conditions in which your complex forms 
crystals.  Feel free to reach out to the National Crystallization Center at HWI 
if you need assistance with finding new conditions (www.getacrystal.org).

Good luck!
Sarah

Sarah EJ Bowman PhD
Associate Investigator | Hauptman-Woodward Medical Research Institute
Director | National High-Throughput Crystallization Center

p: +1 716 898 8623
e: sbowman at hwi.buffalo.edu

Research Webpage<https://hwi.buffalo.edu/scientist-directory/sbowman/>
Crystallization Center Webpage<http://www.getacrystal.org>

Hauptman-Woodward Medical Research Institute
700 Ellicott Street | Buffalo, NY 14203

From: CCP4 bulletin board  on behalf of "Blake, Morgan 
Elizabeth" 
Reply-To: "Blake, Morgan Elizabeth" 
Date: Wednesday, November 22, 2023 at 11:54 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Fragile Crystals

Hello! I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350,
Warning! This message was sent from outside your organization and we were 
unable to verify the sender.

sophospsmartbannerend
Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Fragile Crystals

[David J. Schuller]

There are multiple avenues you could explore.

If you think handling is an issue, you could look into growing and collecting 
with "in situ"  plates.

You could test for diffraction at room temp, or even get a full data set at 
room temp by combining data from multiple crystals. If diffraction is not good 
at room temp, it is probably not going to be better under cryo conditions.

As for cryo, instead of trying cryoprotectant A or cryoprotectant B, you could 
try a mixture of multiple cryoprotectants:

https://pubs.acs.org/doi/abs/10.1021/acs.cgd.5b01692
Use of Multiple Cryoprotectants to Improve Diffraction Quality from Protein 
Crystals
Senda, et al.
https://doi.org/10.1021/acs.cgd.5b01692

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from 
Staphylococcus aureus
JE Sanchez, PG Gross, RW Goetze, RM Walsh Jr, WB Peeples, ZA Wood
Biochemistry 54 (21), 3360-3369

===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu

From: CCP4 bulletin board  on behalf of Blake, Morgan 
Elizabeth 
Sent: Wednesday, November 22, 2023 11:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Fragile Crystals

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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Re: [ccp4bb] Fragile Crystals

[Nicholas Clark]

Hi Morgan,

Have you tried MMS?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157405/

If you can reproducibly grow crystals MMS might give you hits in different
conditions during screening. I’ve used this successfully multiple times for
crystals that wouldn’t optimize with standard methods. We were able to
solve the structure in each case.

Hope this helps.

Best,

Nick Clark

On Wed, Nov 22, 2023 at 11:54 AM Blake, Morgan Elizabeth 
wrote:

> Hello!
>
> I am a PhD student working on a crystallography project to wrap up my
> dissertation research. I have purified a complex of two proteins, and I can
> consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane
> pH 7.5. These crystals have sharp edges and can grow to a large size
> (greater than 0.5 mm), but the crystals seem to be very fragile. When we
> open the drops to harvest the crystals, we have little time to harvest the
> crystals before they crack. When we move the crystals to a cryoprotectant,
> over time they start fracturing. We've tried using different percentages of
> glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no
> success. Needless to say, the crystals do not diffract well, with spot
> patterns that look very streaky/mosaic, which I presume is due to the
> defects that we see in harvesting/handling. We have screened for alternate
> crystallization conditions, but we seem to get the same morphology in other
> conditions. Does anyone have suggestions for additives we could use
> post-crystallization to help stabilize our crystals?
>
> Thanks for your advice!
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
>



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[ccp4bb] Fragile Crystals

[Blake, Morgan Elizabeth]

Hello!

I am a PhD student working on a crystallography project to wrap up my 
dissertation research. I have purified a complex of two proteins, and I can 
consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 
7.5. These crystals have sharp edges and can grow to a large size (greater than 
0.5 mm), but the crystals seem to be very fragile. When we open the drops to 
harvest the crystals, we have little time to harvest the crystals before they 
crack. When we move the crystals to a cryoprotectant, over time they start 
fracturing. We've tried using different percentages of glycerol, ethylene 
glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, 
the crystals do not diffract well, with spot patterns that look very 
streaky/mosaic, which I presume is due to the defects that we see in 
harvesting/handling. We have screened for alternate crystallization conditions, 
but we seem to get the same morphology in other conditions. Does anyone have 
suggestions for additives we could use post-crystallization to help stabilize 
our crystals?

Thanks for your advice!



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