Re: [ccp4bb] sticky crystals

[Jeff Speir]

I second Chris's suggestions.  These have worked well for me in the  
past.  You only need a very thin layer of the grease (i.e. keep  
wiping until its almost completely gone) and it usually has no affect  
on the crystallization.


Jeff


On Jan 27, 2009, at 3:51 PM, Christopher Colbert wrote:


If you have good and bad crystals in the same drop, I've had success
pushing a crummy crystal into a good crystal and having it release  
that

way.

Additionally, once I realized this was going to be a long term  
problem, I
started coating the sitting drop depressions with a thin layer of  
vacuum
grease.  The crystals just slid right off the grease and I never  
saw any

changes in the diffraction data to suggest the grease was giving me
issues.

Chris

On Wed, 28 Jan 2009, Savvas Savvides wrote:


Dear colleagues,

we have been growing crystals of a protein complex  in sitting- 
drop geometry
that stick to the bottom of the drop remarkably well. It's as if  
they are
glued onto the plastic. This makes crystal handling next to  
impossible
without destroying the crystals. We have tried whiskers, loops,  
all kinds of
micro-tools, and pipetting techniques to no avail.  I can say at  
the outset
that we have been unsuccessful in growing these crystals in  
hanging-drops or
at 4 degrees. Deglycosylating the complex also leads to nowhere.  
In fact, we
are only able to get crystals from homogeneously glycosylated  
protein

produced in HEK293S/I- cells.



In the meantime we are playing with the idea of  siliconizing the
sitting-drop depressions to alter the crystal/plate interface.  
But then
again, nucleation events on the plastic  may be the reason we  
are getting
crystals in the first place. We have also thought of trying  
microseeding to
have more control on nucleation issues. Our protein production  
is quite
limiting and forces us to be very selective with our  
experimentation.




Nonetheless,  while we are waiting for fresh material  to  
explore some of
these ideas we would like to make the most out of the crystals  
we have grown
thus far. We would therefore very much appreciate any input/ 
ideas on

manipulating these crystals for data collection.



Best wishes

Savvas






Savvas Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
Email: savvas.savvi...@ugent.be
http://www.lprobe.ugent.be/xray.html







From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On  
Behalf Of

Katarina Moravcevic
Sent: Tuesday, January 27, 2009 10:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo translation



Hi all,

here is a question from a beginner. I have a home source data  
set  that
indexed and scaled in a P2 space group  (a=46.704,b=59.362,  
c=48.783,
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au.  
After failing
to get a MR solution with Phaser I ran the phenix.xtriage which  
showed that
I have a large (89.18) Patterson peak at 0.5 0 0.5 which  
indicates pseudo
translational symmetry. I was wondering if there is anything I  
could do with
this data to get around this problem. Given that I don't have a  
lot of

experience any suggestion/explanation would be fantastic.

Thanks in advance

K





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Christopher L. Colbert, Ph.D.
InstructorPhone: (214) 645  
5944
University of Texas Southwestern Medical Center	  FAX:   (214) 645  
5945

6001 Forest Park Lane
Dallas, TX 75390

Re: [ccp4bb] sticky crystals

[Christian Biertuempfel]

Hi Savvas,
If the very good suggestions you have already got from the ccp4bb do not
help, try crystallization with agarose as an additive. Crystals form
inside the very soft gel and they are hold in place by this meshwork.
So, they are mechanically protected and do not fall down onto the bottom
of the sitting-drop well. A final concentration of 0.1-0.2 % (w/v)
agarose is sufficient. When you harvest a crystal cut generously around
it with a microtool, pick it up (e.g. using a nylon loop) and do not
mind if some agarose comes with it.

for example:

Biertümpfel, C.; Basquin, J.; Suck, D. & Sauter, C.
Crystallization of biological macromolecules using agarose gel.
Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9
PMID: 12351881

Good luck!
christian


Savvas Savvides wrote:
> Dear colleagues,
> 
> we have been growing crystals of a protein complex  in sitting-drop
> geometry that stick to the bottom of the drop remarkably well. It’s as
> if they are glued onto the plastic. This makes crystal handling next to
> impossible without destroying the crystals. We have tried whiskers,
> loops, all kinds of micro-tools, and pipetting techniques to no avail.
>  I can say at the outset that we have been unsuccessful in growing these
> crystals in hanging-drops or at 4 degrees. Deglycosylating the complex
> also leads to nowhere. In fact, we are only able to get crystals from
> homogeneously glycosylated protein produced in HEK293S/I- cells.
> 
>  
> 
>  In the meantime we are playing with the idea of  siliconizing the
> sitting-drop depressions to alter the crystal/plate interface. But then
> again, nucleation events on the plastic  may be the reason we are
> getting crystals in the first place. We have also thought of trying
> microseeding to have more control on nucleation issues. Our protein
> production is quite limiting and forces us to be very selective with our
> experimentation.
> 
>  
> 
> Nonetheless,  while we are waiting for fresh material  to explore some
> of these ideas we would like to make the most out of the crystals we
> have grown thus far. We would therefore very much appreciate any
> input/ideas on manipulating these crystals for data collection.
> 
>  
> 
> Best wishes
> 
> Savvas
> 
>  
> 
>  
> 
> 
> Savvas Savvides
> L-ProBE, Unit for Structural Biology
> Ghent University
> K.L. Ledeganckstraat 35
> 9000 Ghent, BELGIUM
> office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
> Email: savvas.savvi...@ugent.be
> http://www.lprobe.ugent.be/xray.html
> 
>  
> 
>  
> 
>  
> 
> *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of
> *Katarina Moravcevic
> *Sent:* Tuesday, January 27, 2009 10:52 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] pseudo translation
> 
>  
> 
> Hi all,
> 
> here is a question from a beginner. I have a home source data set  that
> indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783,
> alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After
> failing to get a MR solution with Phaser I ran the phenix.xtriage which
> showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which
> indicates pseudo translational symmetry. I was wondering if there is
> anything I could do with this data to get around this problem. Given
> that I don't have a lot of experience any suggestion/explanation would
> be fantastic. 
> 
> Thanks in advance
> 
> K
> 
> 
> 
> *
> 
> E-mail message checked by Spyware Doctor (6.0.0.386)
> Database version: 5.11630
> http://www.pctools.com/spyware-doctor-antivirus/
> 
> *


___

Dr. Christian Biertümpfel
Laboratory of Molecular Biology

NIDDK/National Institutes of Health  phone: +1 301 402 4647
9000 Rockville Pike, Bldg. 5, Rm. B1-03  fax:   +1 301 496 0201
Bethesda, MD 20892-0580
USA
___

Re: [ccp4bb] sticky crystals

[mesters]

Most obvious (maybe you did it already but that is not clear from your 
email), why not try seeding?


- J. -


Savvas Savvides wrote:


Dear colleagues,

we have been growing crystals of a protein complex in sitting-drop 
geometry that stick to the bottom of the drop remarkably well. It’s as 
if they are glued onto the plastic. This makes crystal handling next 
to impossible without destroying the crystals. We have tried whiskers, 
loops, all kinds of micro-tools, and pipetting techniques to no avail. 
I can say at the outset that we have been unsuccessful in growing 
these crystals in hanging-drops or at 4 degrees. Deglycosylating the 
complex also leads to nowhere. In fact, we are only able to get 
crystals from homogeneously glycosylated protein produced in 
HEK293S/I- cells.


In the meantime we are playing with the idea of siliconizing the 
sitting-drop depressions to alter the crystal/plate interface. But 
then again, nucleation events on the plastic may be the reason we are 
getting crystals in the first place. We have also thought of trying 
microseeding to have more control on nucleation issues. Our protein 
production is quite limiting and forces us to be very selective with 
our experimentation.


Nonetheless, while we are waiting for fresh material to explore some 
of these ideas we would like to make the most out of the crystals we 
have grown thus far. We would therefore very much appreciate any 
input/ideas on manipulating these crystals for data collection.


Best wishes

Savvas


Savvas Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
Email: savvas.savvi...@ugent.be
http://www.lprobe.ugent.be/xray.html

*From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf 
Of *Katarina Moravcevic

*Sent:* Tuesday, January 27, 2009 10:52 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] pseudo translation

Hi all,

here is a question from a beginner. I have a home source data set that 
indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, 
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After 
failing to get a MR solution with Phaser I ran the phenix.xtriage 
which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 
which indicates pseudo translational symmetry. I was wondering if 
there is anything I could do with this data to get around this 
problem. Given that I don't have a lot of experience any 
suggestion/explanation would be fantastic.


Thanks in advance

K



*

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Database version: 5.11630
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*



--
Dr. Jeroen R. Mesters
Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
Institut für Biochemie, Universität zu Lübeck
Zentrum für Medizinische Struktur- und Zellbiologie
Ratzeburger Allee 160, D-23538 Lübeck
Tel: +49-451-5004065, Fax: +49-451-5004068
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.selfish-brain.org
Http://www.opticryst.org
--
If you can look into the seeds of time and say
which grain will grow and which will not - speak then to me  (Macbeth)
--

Re: [ccp4bb] sticky crystals

[raz]

Hi All

One more method that I heard about but never tried is to put the plate 
on a sonication bath and to let for a short sonication pulse. That 
should vibrate some liquid under your crystal.

Raz

-- 
 

Raz Zarivach, Ph.D.

Department of Life Sciences and the National Institute of Biotechnology in the 
Negev
Ben-Gurion University of the Negev
POB 653
Zip code 84105
Beer-Sheva
Israel

Home page: http://raz189.tripod.com/
tel: +972-8-646-1999
cell: +972-50-5754808
fax: +972-8-6472970
skype: zarivach
ra...@yahoo.com









  

Re: [ccp4bb] sticky crystals

[Jean-Luc Ferrer]

Hi Savvas,

You can collect data on your crystal still in the drop, on our beamline
(FIP-BM30A at the ESRF) if you are interested. Provided space group is
not P1 We do that routinely.
Let me know if you are interested.

JL

Savvas Savvides wrote:

  
  
  

  
  Dear
colleagues,
  we
have been growing crystals of a protein complex  in
sitting-drop geometry that stick to the bottom of the drop remarkably
well. It’s
as if they are glued onto the plastic. This makes crystal handling next
to
impossible without destroying the crystals. We have tried whiskers,
loops, all
kinds of micro-tools, and pipetting techniques to no avail.  I can say
at
the outset that we have been unsuccessful in growing these crystals in
hanging-drops or at 4 degrees. Deglycosylating the complex also leads
to
nowhere. In fact, we are only able to get crystals from homogeneously
glycosylated
protein produced in HEK293S/I- cells. 
   
   In
the meantime we are playing with the idea of  siliconizing
the sitting-drop depressions to alter the crystal/plate interface. But
then
again, nucleation events on the plastic  may be the reason we are
getting
crystals in the first place. We have also thought of trying
microseeding to have
more control on nucleation issues. Our protein production is quite
limiting and
forces us to be very selective with our experimentation.
   
  Nonetheless,
 while we are waiting for fresh material  to
explore some of these ideas we would like to make the most out of the
crystals
we have grown thus far. We would therefore very much appreciate any
input/ideas
on manipulating these crystals for data collection.
   
  Best
wishes
  Savvas
   
   
  
  
  Savvas
Savvides
  
  L-ProBE,
Unit for Structural Biology
  
  Ghent
University
  
  K.L.
Ledeganckstraat 35
  
  9000
Ghent, BELGIUM
  
  office:
+32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
  
  Email:
savvas.savvi...@ugent.be
  
http://www.lprobe.ugent.be/xray.html
   
   
   
  
  From: CCP4
bulletin board
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarina Moravcevic
  Sent: Tuesday, January 27, 2009 10:52 PM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] pseudo translation
  
   
  Hi all,
  
  here is a question from a beginner. I have a
home
source data set  that indexed and scaled in a P2 space group
 (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0)
with predicted 2mol/au. After failing to get a MR solution with Phaser
I ran
the phenix.xtriage which showed that I have a large (89.18) Patterson
peak at
0.5 0 0.5 which indicates pseudo translational symmetry. I was
wondering if
there is anything I could do with this data to get around this problem.
Given
that I don't have a lot of experience any suggestion/explanation would
be
fantastic. 
  
  
  Thanks in advance
  
  
  K
  
  
  
  
  
  
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-- 
Jean-Luc Ferrer
|---|
|Institut de Biologie Structurale J.P. Ebel CEA/CNRS/UJF|
| 41 rue Jules Horowitz, 38027 Grenoble cedex 1, FRANCE |
|tel.: +33 (0)4-38-78-59-10,  fax : +33 (0)4-38-78-51-22|
|---|

Re: [ccp4bb] sticky crystals

[deliang]

Hi,

I had a similar story like yours.Then I added a drop of 10ul simulated mother 
liquot which contains much higher concentrations of all components in the 
normal mother liquot. Sometimes, the crystals attached to the plastic would 
float to the  surface. If not, take another  10ul, but blew it to the bottom 
plastic with a pippetman back and forth, and some crystals would also leave the 
plastic(But you have to be very careful to do this.)

My friend Jill solved her similar problem by hanging drop setup instead of 
sitting drop.

Good luck.

Deliang


  - Original Message - 
  From: Savvas Savvides 
  To: CCP4BB@JISCMAIL.AC.UK 
  Sent: Tuesday, January 27, 2009 3:05 PM
  Subject: [ccp4bb] sticky crystals


  Dear colleagues,

  we have been growing crystals of a protein complex  in sitting-drop geometry 
that stick to the bottom of the drop remarkably well. It's as if they are glued 
onto the plastic. This makes crystal handling next to impossible without 
destroying the crystals. We have tried whiskers, loops, all kinds of 
micro-tools, and pipetting techniques to no avail.  I can say at the outset 
that we have been unsuccessful in growing these crystals in hanging-drops or at 
4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are 
only able to get crystals from homogeneously glycosylated protein produced in 
HEK293S/I- cells. 

   

   In the meantime we are playing with the idea of  siliconizing the 
sitting-drop depressions to alter the crystal/plate interface. But then again, 
nucleation events on the plastic  may be the reason we are getting crystals in 
the first place. We have also thought of trying microseeding to have more 
control on nucleation issues. Our protein production is quite limiting and 
forces us to be very selective with our experimentation.

   

  Nonetheless,  while we are waiting for fresh material  to explore some of 
these ideas we would like to make the most out of the crystals we have grown 
thus far. We would therefore very much appreciate any input/ideas on 
manipulating these crystals for data collection.

   

  Best wishes

  Savvas

   

   

   
  Savvas Savvides 
  L-ProBE, Unit for Structural Biology 
  Ghent University 
  K.L. Ledeganckstraat 35 
  9000 Ghent, BELGIUM 
  office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
  Email: savvas.savvi...@ugent.be 
  http://www.lprobe.ugent.be/xray.html

   

   

   

  From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of 
Katarina Moravcevic
  Sent: Tuesday, January 27, 2009 10:52 PM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] pseudo translation

   

  Hi all,

  here is a question from a beginner. I have a home source data set  that 
indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783, 
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to 
get a MR solution with Phaser I ran the phenix.xtriage which showed that I have 
a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo 
translational symmetry. I was wondering if there is anything I could do with 
this data to get around this problem. Given that I don't have a lot of 
experience any suggestion/explanation would be fantastic. 

  Thanks in advance

  K





  E-mail message checked by Spyware Doctor (6.0.0.386)
  Database version: 5.11630
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Re: [ccp4bb] sticky crystals

[James Holton]

Suggestions so far have been good ones. However, the MiTeGen microtools kit:
http://mitegen.com/products/microtools/microtools_kit1.shtml

comes with a "MicroSaw", which is a 10-micron thick kapton saw that is 
intended for this purpose. That is, you don't pry the crystal off the 
surface, but rather rest this saw against the surface, bring it over to 
the edge of the stuck crystal and then work it back and forth until you 
have cut underneath the crystal. Did you try this tool?


-James Holton
MAD Scientist


Savvas Savvides wrote:


Dear colleagues,

we have been growing crystals of a protein complex in sitting-drop 
geometry that stick to the bottom of the drop remarkably well. It’s as 
if they are glued onto the plastic. This makes crystal handling next 
to impossible without destroying the crystals. We have tried whiskers, 
loops, all kinds of micro-tools, and pipetting techniques to no avail. 
I can say at the outset that we have been unsuccessful in growing 
these crystals in hanging-drops or at 4 degrees. Deglycosylating the 
complex also leads to nowhere. In fact, we are only able to get 
crystals from homogeneously glycosylated protein produced in 
HEK293S/I- cells.


In the meantime we are playing with the idea of siliconizing the 
sitting-drop depressions to alter the crystal/plate interface. But 
then again, nucleation events on the plastic may be the reason we are 
getting crystals in the first place. We have also thought of trying 
microseeding to have more control on nucleation issues. Our protein 
production is quite limiting and forces us to be very selective with 
our experimentation.


Nonetheless, while we are waiting for fresh material to explore some 
of these ideas we would like to make the most out of the crystals we 
have grown thus far. We would therefore very much appreciate any 
input/ideas on manipulating these crystals for data collection.


Best wishes

Savvas


Savvas Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
Email: savvas.savvi...@ugent.be
http://www.lprobe.ugent.be/xray.html

*From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf 
Of *Katarina Moravcevic

*Sent:* Tuesday, January 27, 2009 10:52 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] pseudo translation

Hi all,

here is a question from a beginner. I have a home source data set that 
indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, 
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After 
failing to get a MR solution with Phaser I ran the phenix.xtriage 
which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 
which indicates pseudo translational symmetry. I was wondering if 
there is anything I could do with this data to get around this 
problem. Given that I don't have a lot of experience any 
suggestion/explanation would be fantastic.


Thanks in advance

K



*

E-mail message checked by Spyware Doctor (6.0.0.386)
Database version: 5.11630
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*

Re: [ccp4bb] sticky crystals

[Christopher Colbert]

If you have good and bad crystals in the same drop, I've had success 
pushing a crummy crystal into a good crystal and having it release that 
way.

Additionally, once I realized this was going to be a long term problem, I 
started coating the sitting drop depressions with a thin layer of vacuum 
grease.  The crystals just slid right off the grease and I never saw any 
changes in the diffraction data to suggest the grease was giving me 
issues.

Chris

On Wed, 28 Jan 2009, Savvas Savvides wrote:

>>>Dear colleagues,
>>>
>>>we have been growing crystals of a protein complex  in sitting-drop geometry
>>>that stick to the bottom of the drop remarkably well. It's as if they are
>>>glued onto the plastic. This makes crystal handling next to impossible
>>>without destroying the crystals. We have tried whiskers, loops, all kinds of
>>>micro-tools, and pipetting techniques to no avail.  I can say at the outset
>>>that we have been unsuccessful in growing these crystals in hanging-drops or
>>>at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we
>>>are only able to get crystals from homogeneously glycosylated protein
>>>produced in HEK293S/I- cells. 
>>>
>>> 
>>>
>>> In the meantime we are playing with the idea of  siliconizing the
>>>sitting-drop depressions to alter the crystal/plate interface. But then
>>>again, nucleation events on the plastic  may be the reason we are getting
>>>crystals in the first place. We have also thought of trying microseeding to
>>>have more control on nucleation issues. Our protein production is quite
>>>limiting and forces us to be very selective with our experimentation.
>>>
>>> 
>>>
>>>Nonetheless,  while we are waiting for fresh material  to explore some of
>>>these ideas we would like to make the most out of the crystals we have grown
>>>thus far. We would therefore very much appreciate any input/ideas on
>>>manipulating these crystals for data collection.
>>>
>>> 
>>>
>>>Best wishes
>>>
>>>Savvas
>>>
>>> 
>>>
>>> 
>>>
>>> 
>>>Savvas Savvides 
>>>L-ProBE, Unit for Structural Biology 
>>>Ghent University 
>>>K.L. Ledeganckstraat 35 
>>>9000 Ghent, BELGIUM 
>>>office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
>>>Email: savvas.savvi...@ugent.be 
>>>http://www.lprobe.ugent.be/xray.html
>>>
>>> 
>>>
>>> 
>>>
>>> 
>>>
>>>From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
>>>Katarina Moravcevic
>>>Sent: Tuesday, January 27, 2009 10:52 PM
>>>To: CCP4BB@JISCMAIL.AC.UK
>>>Subject: [ccp4bb] pseudo translation
>>>
>>> 
>>>
>>>Hi all,
>>>
>>>here is a question from a beginner. I have a home source data set  that
>>>indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783,
>>>alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing
>>>to get a MR solution with Phaser I ran the phenix.xtriage which showed that
>>>I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo
>>>translational symmetry. I was wondering if there is anything I could do with
>>>this data to get around this problem. Given that I don't have a lot of
>>>experience any suggestion/explanation would be fantastic. 
>>>
>>>Thanks in advance
>>>
>>>K
>>>
>>>
>>>
>>>
>>>
>>>E-mail message checked by Spyware Doctor (6.0.0.386)
>>>Database version: 5.11630
>>>http://www.pctools.com/en/spyware-doctor-antivirus/
>>>

Christopher L. Colbert, Ph.D.
InstructorPhone: (214) 645 5944
University of Texas Southwestern Medical Center   FAX:   (214) 645 5945
6001 Forest Park Lane
Dallas, TX 75390

Re: [ccp4bb] sticky crystals

[Artem Evdokimov]

Hi,

 

I had this exact problem before. The method below worked for me:

 

Take a sturdy needle (like one of the microneedles from a Hampton kit or a
very thin syringe needle) and (while observing the whole thing under a
scope) stick the needle into the plastic a bit away from the crystal. Push
hard. If you're using polarizers, you may b abe to visualize the stress
forces in the plastic by the shifting of the colors. The basic idea here is
to stress the plastic under the crystal w/o touching the crystal in any way.
In my case the bloody things just popped off. Sometimes you have to push
quite hard, and to wiggle the needle a bit - and beware, they can slip and
ruin your drop. But this really worked quite well!

 

Artem

 

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Savvas
Savvides
Sent: Tuesday, January 27, 2009 6:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] sticky crystals

 

Dear colleagues,

we have been growing crystals of a protein complex  in sitting-drop geometry
that stick to the bottom of the drop remarkably well. It's as if they are
glued onto the plastic. This makes crystal handling next to impossible
without destroying the crystals. We have tried whiskers, loops, all kinds of
micro-tools, and pipetting techniques to no avail.  I can say at the outset
that we have been unsuccessful in growing these crystals in hanging-drops or
at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we
are only able to get crystals from homogeneously glycosylated protein
produced in HEK293S/I- cells. 

 

 In the meantime we are playing with the idea of  siliconizing the
sitting-drop depressions to alter the crystal/plate interface. But then
again, nucleation events on the plastic  may be the reason we are getting
crystals in the first place. We have also thought of trying microseeding to
have more control on nucleation issues. Our protein production is quite
limiting and forces us to be very selective with our experimentation.

 

Nonetheless,  while we are waiting for fresh material  to explore some of
these ideas we would like to make the most out of the crystals we have grown
thus far. We would therefore very much appreciate any input/ideas on
manipulating these crystals for data collection.

 

Best wishes

Savvas

 

 

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html

 

 

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Katarina Moravcevic
Sent: Tuesday, January 27, 2009 10:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo translation

 

Hi all,

here is a question from a beginner. I have a home source data set  that
indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783,
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing
to get a MR solution with Phaser I ran the phenix.xtriage which showed that
I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo
translational symmetry. I was wondering if there is anything I could do with
this data to get around this problem. Given that I don't have a lot of
experience any suggestion/explanation would be fantastic. 

Thanks in advance

K





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Re: [ccp4bb] sticky crystals

[Jim Pflugrath]

Put a small piece of dry ice on the opposite side of the plastic from the 
crystal.  Perhaps the difference in thermal expansive coefficient will let 
the crystal(s) break away.  Don't overdo it though.  This is a trick that 
Gary Gilliland taught me.


Jim

On Wed, 28 Jan 2009, Savvas Savvides wrote:


Dear colleagues,

we have been growing crystals of a protein complex  in sitting-drop geometry
that stick to the bottom of the drop remarkably well. It's as if they are
glued onto the plastic. This makes crystal handling next to impossible
without destroying the crystals. We have tried whiskers, loops, all kinds of
micro-tools, and pipetting techniques to no avail.  I can say at the outset
that we have been unsuccessful in growing these crystals in hanging-drops or
at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we
are only able to get crystals from homogeneously glycosylated protein
produced in HEK293S/I- cells.



In the meantime we are playing with the idea of  siliconizing the
sitting-drop depressions to alter the crystal/plate interface. But then
again, nucleation events on the plastic  may be the reason we are getting
crystals in the first place. We have also thought of trying microseeding to
have more control on nucleation issues. Our protein production is quite
limiting and forces us to be very selective with our experimentation.



Nonetheless,  while we are waiting for fresh material  to explore some of
these ideas we would like to make the most out of the crystals we have grown
thus far. We would therefore very much appreciate any input/ideas on
manipulating these crystals for data collection.



Best wishes

Savvas






Savvas Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
Email: savvas.savvi...@ugent.be
http://www.lprobe.ugent.be/xray.html







From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Katarina Moravcevic
Sent: Tuesday, January 27, 2009 10:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] pseudo translation



Hi all,

here is a question from a beginner. I have a home source data set  that
indexed and scaled in a P2 space group  (a=46.704,b=59.362, c=48.783,
alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing
to get a MR solution with Phaser I ran the phenix.xtriage which showed that
I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo
translational symmetry. I was wondering if there is anything I could do with
this data to get around this problem. Given that I don't have a lot of
experience any suggestion/explanation would be fantastic.

Thanks in advance

K





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