Re: [ccp4bb] Cryo

[Nicolas Soler]

Hi Vitul,

Having a final concentration of 2M for lithium sulphate worked for me 
(from a 2.5M stock solution).


Nicolas

On 09/10/2014 10:27, vitul jain wrote:

Hello everybody,

can anyone please suggest a good Cryo for condition having 0.8 M 
Lithium sulphate and 0.1 M  Sodium acetate 4.6.


Thanks in advance

Vitul


--
Vitul Jain
PhD student
C/O Dr. Amit Sharma
Structural and Computational Biology lab
International Center for Genetic Engineering and Biotechnology
Aruna Asaf Ali road, New Delhi
110067.

Mb. no: +91-9818004350, 8860942543
E-mail: vituljain...@gmail.com  / 
vi...@icgeb.res.in 



--
Nicolas Soler
Roger Williams group
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge CB2 0QH
United Kingdom
phone : +44(0) 1223 26 76 20
mail : nso...@mrc-lmb.cam.ac.uk

Re: [ccp4bb] Cryo

[Enrico Stura]

 Vitul,

0.8 M  Lithium sulphate is not excessively high salt (a 2X precipitant is  
possible)

You have many options available:
2M for lithium sulphate is one of them see:
http://pubs.acs.org/doi/full/10.1021/cg301531f
for various combinations of mixed cryoprotectants.

Enrico.




.On Thu, 09 Oct 2014 11:55:49 +0200, Nicolas Soler  
 wrote:



Hi Vitul,

Having a final concentration of 2M for lithium sulphate worked for me
(from a 2.5M stock solution).

Nicolas

On 09/10/2014 10:27, vitul jain wrote:

Hello everybody,

can anyone please suggest a good Cryo for condition having 0.8 M
Lithium sulphate and 0.1 M  Sodium acetate 4.6.

Thanks in advance

Vitul


--
Vitul Jain
PhD student
C/O Dr. Amit Sharma
Structural and Computational Biology lab
International Center for Genetic Engineering and Biotechnology
Aruna Asaf Ali road, New Delhi
110067.

Mb. no: +91-9818004350, 8860942543
E-mail: vituljain...@gmail.com  /
vi...@icgeb.res.in 






--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryo-cooling

[Brandao-Neto, J (Jose)]

 does anybody have a reference for that paper (or was it a poster?) by robert 
thorne on systematic investigations about the cooling process? are there 
similar studies with propane? isn't propane a pain for transporting samples 
because of the regulations?
 
regards,
jose
 

Systems Biology Talks at Diamond (SR User Meeting 2007) 
http://systemsbiology07.wordpress.com/ 
<https://outlook.rl.ac.uk/exchweb/bin/redir.asp?URL=http://systemsbiology07.wordpress.com/>
 

Jose Ribeiro Brandao-Neto, MPhil
Beamline Scientist - I04-1
Macromolecular Crystallography Group
 
Diamond Light Source Ltd
Diamond House DR1.78
Harwell Science and Innovation Campus
RAL, Chilton, Didcot
Oxfordshire
OX11 0DE
United Kingdom
 
tel: +44 (0)1235 778506
 +44 (0)1235 778000
fax: +44 (0)1235 778448
 
[EMAIL PROTECTED]
http://www.diamond.ac.uk 
<https://outlook.rl.ac.uk/exchweb/bin/redir.asp?URL=http://www.diamond.ac.uk/> 
 
 



From: CCP4 bulletin board on behalf of Jim Pflugrath
Sent: Fri 06/06/2008 19:24
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo-cooling, was: Re: [ccp4bb] crystallisation and 
mosaicity



> Jim,
>
> The fact that liquid propane can exist at a range of temperatures is actually
> a MAJOR advantage.  While you must ensure that the temperature of the liquid
> propane is just above its own freezing point, the very high boiling point of
> propane ensures that there is liquid-to-solid contact with your sample for
> good heat transfer.  When the cryogen boils, there is a gas-to-solid contact,
> slower cooling, and a greater chance of ice crystal formation.
>

Yes, I agree with the theory, but I also I believe that if you quickly
plunge your crystal into liquid nitrogen, that the high velocity through
the liquid presents fresh liquid nitrogen as you go and the gas-to-solid
contact is neglible.  I think speed and having no cold-gas-layer above the
liquid cryogen are the main points of the Warkentin et al. paper cited
previously.  I have observed many slow people freezing crystals instead of
flash-cooling them.

But for some crystals flash-cooling is better at temperatures higher than
in the 77 K to 100 K regime for unknown reasons.  This can be one reason
why flash-cooling in the gas stream occassionally is better.  It is also
why liquid propane worked for Raji E. and Karolin L.: they were using a
temperature above the freezing point of propane.

If you are having problems with flash-cooling, go ahead and try propane
(my preference is CF4 over propane though).  And a trick we learned from
the University of Cambridge is to fill a balloon with the gas, put the
balloon opening over a 15 ml Falcon tube and then condense/freeze the gas
in the tube for pouring into vials later.  This prevents wasting gas by
bubbling through a coil held in liquid nitrogen.  This is just one of
techniques described in the PDF I mentioned.

It is also not a bad idea to practice flash-cooling on lysozyme or
thaumatin crystals and get good at it before working with your own
precious crystals.

Jim


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Re: [ccp4bb] cryo-cooling

[Kay Diederichs]

Jose,

check out the 4 papers by R. Thorne listed at
http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html

HTH,

Kay
--
Kay Diederichs  http://strucbio.biologie.uni-konstanz.de
email: [EMAIL PROTECTED]  Tel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz



smime.p7s
Description: S/MIME Cryptographic Signature

Re: [ccp4bb] cryo-cooling

[Oganesyan, Vaheh]

I apologize for using BB to address a person (Ethan Merritt, U. of Washington). 
His e-mail address mentioned on TLSMD page is not operational.


Good morning Ethan,

Few months ago I asked you if carbohydrates can be recognized by TLSMD and 
appropriate calculations can be included in on-line version of TLSMD. I'm 
writing to you to find out if there was any progress in that direction and how 
to make carbohydrates to be recognized.

Regards,

Vaheh Oganesyan

___
Vaheh

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Kay Diederichs
Sent: Saturday, June 07, 2008 7:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo-cooling

Jose,

check out the 4 papers by R. Thorne listed at
http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html

HTH,

Kay
-- 
Kay Diederichs  http://strucbio.biologie.uni-konstanz.de
email: [EMAIL PROTECTED]  Tel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz




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Re: [ccp4bb] Cryo-protectant

[Tim Gruene]

Hallo,

there is quite a large number of cryo-protectants other than MPD or 
glycerol. If I remember correctly, the Methods of Enzymology, (176) list 
some of the, and you might check the recipes of the Hampton cryo screen.


As suggested by Li, you can try oils. You can use small sugars (fructose, 
glucose...), PEG400, or 1,6-butanediol (maybe this was 2,5-butanediol) and 
mixtures of the above, e.g. 15%PEG400 + 5% glycerol etc.


It also matters how you freeze the crystals - whether you freeze them in 
the nitrogen stream at the X-ray machine or dip them into a container with 
liquid nitrogen (I prefer the latter)


Also, you can
 - dip them directly into the cryo solution
 - move them step by step to higher concentrations and hereby observe the
   crystals' health under the microscope
 - transport the crystals with a pipette rather than using the cryo-loop.
   this techniques reduces their exposure to air and exsiccation.

etc. pp

With an inhouse machine you can also try a measurement at room temperature 
in a capillary.


Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A


On Fri, 24 Apr 2009, Liew Chong Wai wrote:


Hi all,
 
Good day
I used MPD as a cryoprotectant (20%, 30%) for my crystal. However, there
is no diffraction signal at all. Without the MPD cryo, i still manage to
get 5angstrom, but it has very strong ice-ring signal. I used glycerol
(15%, 20%, 25% and 30%) before, but it cracked the crystal.
 
Please advice.
 
Thanks 
 
Liew
 

Re: [ccp4bb] Cryo-protectant

[Roger Rowlett]

I usually try the following, in order:

1. Transfer crystals to mother liquor + 30% glycerol or ethylene glycol
(sometimes lower depending on crystallization solution). This did not
work for you.
2. Transfer crystals to mother liquor + 30% glucose (or try sequential
soaks in M.L. + 15% and then 30% glucose. Just a few sec/min is usually
enough). Glucose or other sugars often work. when glycerol or EG fails.
3. Try the "no-fail" in situ cryo method, which is a gradual buildup of
cryoprotectant. See
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo . This
is very gentle, and often works when #1 and #2 does not, but in our
hands nearly always increases mosaicity. (But mosaic is better than no
diffraction.)
4. Try dragging crystals though paratone-N to remove surface water from
the crystal. This actually nearly always works for us, but is more
fussy than #1 or #2, and it is easier to damage crystals during
manipulation because of the viscosity of the oil.

I normally plunge protected crystals into LN2 after mounting.

Ice rings are a good indication of poor cryoprotection, but lack of
diffraction could just be your crystals, too. For our latest dataset,
we just sorted through 38 crystals until we found a good one. The key,
as it turned out, is that all of our beautiful large crystals were
apparently difficult to visualize disordered stacks of plates (we
didn't notice this until some fractured during cryo-soaks) whereas some
of the small crystals were actually single crystals. We selected a
decently diffracting small one and took lng frames to get a
good data set.

Cheers,

-- 

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@mail.colgate.edu


Liew Chong Wai wrote:

  
  
  
  Hi all, 
   
  Good day
  I used MPD as a
cryoprotectant (20%, 30%) for my crystal. However, there is no
diffraction signal at all. Without the MPD cryo, i still manage to get
5angstrom, but it has very strong ice-ring signal. I used glycerol
(15%, 20%, 25% and 30%) before, but it cracked the crystal. 
   
  Please advice.
   
  Thanks 
   
  
  
  Liew
   
  

Re: [ccp4bb] Cryo-protectant

[Ed Pozharski]

What is the crystallization condition?

On Fri, 2009-04-24 at 11:46 +0800, Liew Chong Wai wrote:
> Hi all, 
>  
> Good day
> I used MPD as a cryoprotectant (20%, 30%) for my crystal. However,
> there is no diffraction signal at all. Without the MPD cryo, i still
> manage to get 5angstrom, but it has very strong ice-ring signal. I
> used glycerol (15%, 20%, 25% and 30%) before, but it cracked the
> crystal. 
>  
> Please advice.
>  
> Thanks 
>  
> Liew
>  
-- 
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

Re: [ccp4bb] Cryo-protectant

[Liew Chong Wai]

Hi all
 
Thanks for your precious suggestions and ideas. 
The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 
35% PEG3350
Now, my crystal seem ok in 20% ethylene glycol, but only after a couple minutes 
of dehydration at room temperature. For sure, i will try other cryoprotectant 
that was suggested here. 
I just wondering why MPD kills the crystal.
Many thanks
 
LIEW
 

 

Re: [ccp4bb] Cryo-protectant

[Andy Torelli]

Hi Liew,

	There have already been some very good suggestions.  I agree with Tim 
Gruene that a great starting point is to test the diffraction properties 
of your crystal at room temperature.  This can serve as a baseline for 
comparing/evaluating cryoprotecting agents and methods.


	You can also test your potential cryoprotection solutions to see if 
they freeze clear or even take a few X-ray snapshots of them to confirm 
there are no ice rings.


	There are lots of publications that can be helpful.  One very helpful 
reference is:

Garman, E.F. and Doublie, S.
Cryocooling of Macromolecular Crystals: Optimisation Methods.
Methods in Enzymology (2003) 368, 188-216.

	Be aware that, as mentioned previously, the method for freezing can 
make a difference (e.g. freezing in cold-stream vs. plunging in 
nitrogen).  An obvious difference is different cooling rates (you can 
find references for this) or less obvious reasons, for example 
differences in dehydration that occur during longer/shorter transfer 
through air from drop to cold-source for either method.


Finally, you can check out the database for cryoprotecting solutions:
http://idb.exst.jaxa.jp/db_data/protein/search-e.php

Good luck,
-Andy

--

=
Andrew T. Torelli Ph.D.
Postdoctoral Associate
Laboratory of Steven E. Ealick
Department of Chemistry and Chemical Biology
Cornell University
=

On 4/24/2009 10:44 AM, Liew Chong Wai wrote:

Hi all
 
Thanks for your precious suggestions and ideas.
The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M 
MgCl.6H2O, 35% PEG3350
Now, my crystal seem ok in 20% ethylene glycol, but only after a couple 
minutes of dehydration at room temperature. For sure, i will try other 
cryoprotectant that was suggested here.

I just wondering why MPD kills the crystal.
Many thanks
 
*LIEW*
 

 







--

=
Andrew T. Torelli Ph.D.
Postdoctoral Associate
Laboratory of Steven E. Ealick
Department of Chemistry and Chemical Biology
Cornell University
=

Re: [ccp4bb] Cryo-protectant

[Joe]

Title: Re: [ccp4bb] Cryo-protectant





First of all, you need to find out if crystals you grew have similar
quality before you conclude poor diffraction is due to cryoprotectant
solutions. As others have suggested, you can test crystals using
capillary mounting method. Second, there are a lot more cryoprotectant
agents out there you can try. Not sure why you sticked to MPD, which is
not even present in your crystallization condition. 

One of protein crystallized in almost the same condition as yours. What
I did is just simply increasing % PEG3350, in addition to 10%-15%
increment of every other ingredients (your protein buffer + well
solution). I also introduced 5% glycerol to bring down % PEG3350. You
can play around % PEG3350 and % glycerol to find a  fine combination
that is cryo-clear and makes you crystals happy. 

Joe

Liew Chong Wai wrote:

  
  
  
  
  Hi all
   
  Thanks for your
precious suggestions and ideas. 
  The crystallization buffer
condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350
  Now, my crystal seem ok in
20% ethylene glycol, but only after a couple minutes of dehydration at
room temperature. For sure, i will try other cryoprotectant that was
suggested here. 
  I just wondering why MPD
kills the crystal.
  Many thanks
   
  
  
  LIEW
   
  
  
 
  
  
  
  
  
  

Re: [ccp4bb] Cryo-protectant

[Ho-Leung Ng]

Your mother liquor is already very close to being a cryoprotectant. I
think adding 5% ethylene glycol, glycerol, etc. is enough. Too much
may damage your crystals. In addition, you can also try:

1. Grow new crystals in your current crystallization condition + 5%
cryoprotectant.

2. Increase the PEG in your reservoir to 40% and allow vapor diffusion
to gently dehydrate your crystals to allow direct freezing.


ho
UC Berkeley

Re: [ccp4bb] cryo-EM

[Brad Bennett]

Yes, but the technique is still in its infancy stage.

DOI: 10.1016/j.sbi.2014.03.004

Cheers-
Brad


On Fri, Aug 29, 2014 at 6:34 AM, rana ibd <
0285de88044a-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear CCP4
> I wanted to ask has anyone tested 3D protein structure by cryo-electron
> microscopy? what is the suitable concentration required for this procedure
> Best Regards
> Rana
>

Re: [ccp4bb] cryo-EM

[Marcus Fislage]

Hi Rana,

in contrast to Brads answer, and in case I understand you well, Cryo  
EM is not in ts infancy stage (as long as you do not talk about Cryo  
EM on crystals.

For cryo EM you need only several ul of a nM solution.
I can always suggest you the 3dem bb. There are of course still  
limitataions on the size of protein target and resolution, but those  
are slowly falling.


Some reading material
http://www.ncbi.nlm.nih.gov/pubmed/23181775
http://www.ncbi.nlm.nih.gov/pubmed/25071206


Cheers
Marcus

Quoting Brad Bennett :


Yes, but the technique is still in its infancy stage.

DOI: 10.1016/j.sbi.2014.03.004

Cheers-
Brad


On Fri, Aug 29, 2014 at 6:34 AM, rana ibd <
0285de88044a-dmarc-requ...@jiscmail.ac.uk> wrote:


Dear CCP4
I wanted to ask has anyone tested 3D protein structure by cryo-electron
microscopy? what is the suitable concentration required for this procedure
Best Regards
Rana







--
Marcus Fislage, PhD

Howard Hughes Medical Institute (HHMI)
Columbia University
Department of Biochemistry and Biophysics
Lab of Joachim Frank
New York, NY

Phone: 212.305.9524
Fax: 212.305.9500

Re: [ccp4bb] cryo-EM

[Andy Sen]

Dear Rana,

Brad's post compelled me to reply to  you. The Cryo EM field is well into its 
pro stage. 

If you can be specific about the MolWt, size and if it makes any complex, then 
much precise and helpful answers to your question can be provided. 

Best 

Anindito Sen Ph.D
JeoL 

> On 29/08/2014, at 11:31 pm, Marcus Fislage  wrote:
> 
> Hi Rana,
> 
> in contrast to Brads answer, and in case I understand you well, Cryo EM is 
> not in ts infancy stage (as long as you do not talk about Cryo EM on crystals.
> For cryo EM you need only several ul of a nM solution.
> I can always suggest you the 3dem bb. There are of course still limitataions 
> on the size of protein target and resolution, but those are slowly falling.
> 
> Some reading material
> http://www.ncbi.nlm.nih.gov/pubmed/23181775
> http://www.ncbi.nlm.nih.gov/pubmed/25071206
> 
> 
> Cheers
> Marcus
> 
> Quoting Brad Bennett :
> 
>> Yes, but the technique is still in its infancy stage.
>> 
>> DOI: 10.1016/j.sbi.2014.03.004
>> 
>> Cheers-
>> Brad
>> 
>> 
>> On Fri, Aug 29, 2014 at 6:34 AM, rana ibd <
>> 0285de88044a-dmarc-requ...@jiscmail.ac.uk> wrote:
>> 
>>> Dear CCP4
>>> I wanted to ask has anyone tested 3D protein structure by cryo-electron
>>> microscopy? what is the suitable concentration required for this procedure
>>> Best Regards
>>> Rana
> 
> 
> 
> -- 
> Marcus Fislage, PhD
> 
> Howard Hughes Medical Institute (HHMI)
> Columbia University
> Department of Biochemistry and Biophysics
> Lab of Joachim Frank
> New York, NY
> 
> Phone: 212.305.9524
> Fax: 212.305.9500

Re: [ccp4bb] cryo-EM

[Brad Bennett]

Hi all-
I interpreted the original question, wrongly so, as an inquiry into using
cryoEM for 3D *crystallography, *which is very new, and not general 3D
structure determination, which of course has been utilized for the last few
decades. My apologies! Yes, there are plenty of success stories out there
for using cryoEM to solve 3D structures, especially recent efforts with
single particle analysis and the use of direct detectors.

Indeed, in my experience, you only need a few uL of a modest concentration
to perform cryoEM experiments; however, size, shape, symmetry, and
dispersity of the sample as well as buffer composition are major variables
in whether you have success or not.

Good luck!
Brad


On Fri, Aug 29, 2014 at 11:06 AM, Andy Sen  wrote:

> Dear Rana,
>
> Brad's post compelled me to reply to  you. The Cryo EM field is well into
> its pro stage.
>
> If you can be specific about the MolWt, size and if it makes any complex,
> then much precise and helpful answers to your question can be provided.
>
> Best
>
> Anindito Sen Ph.D
> JeoL
>
> > On 29/08/2014, at 11:31 pm, Marcus Fislage  wrote:
> >
> > Hi Rana,
> >
> > in contrast to Brads answer, and in case I understand you well, Cryo EM
> is not in ts infancy stage (as long as you do not talk about Cryo EM on
> crystals.
> > For cryo EM you need only several ul of a nM solution.
> > I can always suggest you the 3dem bb. There are of course still
> limitataions on the size of protein target and resolution, but those are
> slowly falling.
> >
> > Some reading material
> > http://www.ncbi.nlm.nih.gov/pubmed/23181775
> > http://www.ncbi.nlm.nih.gov/pubmed/25071206
> >
> >
> > Cheers
> > Marcus
> >
> > Quoting Brad Bennett :
> >
> >> Yes, but the technique is still in its infancy stage.
> >>
> >> DOI: 10.1016/j.sbi.2014.03.004
> >>
> >> Cheers-
> >> Brad
> >>
> >>
> >> On Fri, Aug 29, 2014 at 6:34 AM, rana ibd <
> >> 0285de88044a-dmarc-requ...@jiscmail.ac.uk> wrote:
> >>
> >>> Dear CCP4
> >>> I wanted to ask has anyone tested 3D protein structure by cryo-electron
> >>> microscopy? what is the suitable concentration required for this
> procedure
> >>> Best Regards
> >>> Rana
> >
> >
> >
> > --
> > Marcus Fislage, PhD
> >
> > Howard Hughes Medical Institute (HHMI)
> > Columbia University
> > Department of Biochemistry and Biophysics
> > Lab of Joachim Frank
> > New York, NY
> >
> > Phone: 212.305.9524
> > Fax: 212.305.9500
>

Re: [ccp4bb] cryo-EM

[Anindito Sen]

Dear Rana,

It appears that you have helical biopolymers formed by the proteins. Given the 
size of 630 kDa as the unit cell or the asymmetric unit forming the biopolymer 
you have a fair chance of gaining insight to the proteins. Using cryo EM  you 
have to collect a sizeable amount of data , followed by computing Fourier 
Transform of the filaments. Using the transform you have to index and determine 
the Axial Rise and turn angle of the filaments. You can then choose any program 
available freely online to calculate the density map of the filaments, thus the 
proteins. You may want to look in 

http://3dem.ucsd.edu/software.shtm

under Helices.

I will recommend  you to use 
BSOFT and FreAlign

www.bsoft.ws
http://grigoriefflab.janelia.org/frealign

Combination of these two yield pretty good results.Keep in mind that such a 
work is a complete project in itself. 

Best wishes





Anindito Sen. Ph.D
Scientist & Application Specialist in Biological Sciences
JEOL LTD.
13F, Ohtemachi Nomura Bldg 
2-1-1 Ohtemachi Chiyoda-Ku. Tokyo, Japan
Tel: +81-3-6262-3563
Fax: +81-3-6262-3577

www.jeol.com




On Aug 30, 2014, at 1:29 AM, rana ibd  wrote:

> 
> Dear Anindito
> Thank you for your reply and I apologize for writing the wrong name, I work 
> on the HBx proteins which contain molecular weights of (56, 57, 59)kDa 
> including  a tag, but they are all polymers and in size exclusion 
> chromatography for the three proteins give a size of 630kDa
> Best Regards
> Rana
> 
> From: Andy Sen 
> To: CCP4BB@JISCMAIL.AC.UK 
> Sent: Friday, August 29, 2014 5:06 PM
> Subject: Re: [ccp4bb] cryo-EM
> 
> Dear Rana,
> 
> Brad's post compelled me to reply to  you. The Cryo EM field is well into its 
> pro stage. 
> 
> If you can be specific about the MolWt, size and if it makes any complex, 
> then much precise and helpful answers to your question can be provided. 
> 
> Best 
> 
> Anindito Sen Ph.D
> JeoL 
> 
> > On 29/08/2014, at 11:31 pm, Marcus Fislage  wrote:
> > 
> > Hi Rana,
> > 
> > in contrast to Brads answer, and in case I understand you well, Cryo EM is 
> > not in ts infancy stage (as long as you do not talk about Cryo EM on 
> > crystals.
> > For cryo EM you need only several ul of a nM solution.
> > I can always suggest you the 3dem bb. There are of course still 
> > limitataions on the size of protein target and resolution, but those are 
> > slowly falling.
> > 
> > Some reading material
> > http://www.ncbi.nlm.nih.gov/pubmed/23181775
> > http://www.ncbi.nlm.nih.gov/pubmed/25071206
> > 
> > 
> > Cheers
> > Marcus
> > 
> > Quoting Brad Bennett :
> > 
> >> Yes, but the technique is still in its infancy stage.
> >> 
> >> DOI: 10.1016/j.sbi.2014.03.004
> >> 
> >> Cheers-
> >> Brad
> >> 
> >> 
> >> On Fri, Aug 29, 2014 at 6:34 AM, rana ibd <
> >> 0285de88044a-dmarc-requ...@jiscmail.ac.uk> wrote:
> >> 
> >>> Dear CCP4
> >>> I wanted to ask has anyone tested 3D protein structure by cryo-electron
> >>> microscopy? what is the suitable concentration required for this procedure
> >>> Best Regards
> >>> Rana
> > 
> > 
> > 
> > -- 
> > Marcus Fislage, PhD
> > 
> > Howard Hughes Medical Institute (HHMI)
> > Columbia University
> > Department of Biochemistry and Biophysics
> > Lab of Joachim Frank
> > New York, NY
> > 
> > Phone: 212.305.9524
> > Fax: 212.305.9500
> 

Re: [ccp4bb] cryo condition

[Bert Van-Den-Berg]

How long are you cryoprotecting for?
You don't really need more than a few seconds. If I have sensitive crystals i 
tend to just "swipe" them slowly through the final CP solution and often that 
works. If I would leave them in the CP solution for more than say 30 seconds 
the crystals would behave like you describe.

GL Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Monday, May 04, 2015 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo condition

Hello everyone

Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:

G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.

Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.

Your suggestions will be of immense help

Thanks in advance
--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] cryo condition

[David Briggs]

If in doubt, try dragging the crystal through a 1:1 mix of Paratone-N and
Mineral oil until most or all the mother liquor from surrounding the
crystal has been removed.

There are more tips here:

http://hamptonresearch.com/tip_detail.aspx?id=99

Good luck!

D

On Mon, 4 May 2015 19:45 Faisal Tarique  wrote:

> Hello everyone
>
> Can anybody suggest me a cryo condition for a crystal obtained in
> MIDAS screen of Molecular Dimension:
>
> G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0
>
> G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0
>
> Crystals are in beautiful cuboid shaped but all sorts of PEG
> combinations and Glycerol formulation failed to prevent it from
> cracking and dissolving.
>
> Has anybody faced a similar situation as mentioned above and what
> precaution was taken to prevent it from cracking or dissolving.
>
> Your suggestions will be of immense help
>
> Thanks in advance
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>

Re: [ccp4bb] cryo condition

[Roger Rowlett]

We rarely use glycerol anymore, because it seems to fail so often for 
many of our current proteins. Try glucose, 25-30%. This is most 
conveniently done by weighing 125-150 mg of glucose in a microcentrifuge 
tube, then addding well solution to the 0.5 mL mark and mixing until 
completely dissolved. Then you can try dunking crystals in the cryo 
solution, or, you can try the "no-fail" method (which does fail on 
occasion) of cryoprotecting directly in the crystallization drop by slow 
addition of the cryoprotectant. See 
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals. 
We have often found the slow addition of a glucose cryoprotectant works 
for fragile, high solvent content crystals that are prone to cracking 
under osmotic stress.


Other alternatives include high concentrations of sodium malonate 
(1-2M), or high concentrations of sodium formate (I think around 4 M?). 
These could also be introduced gradually if required.


Good luck!

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 5/4/2015 2:43 PM, Faisal Tarique wrote:

Hello everyone

Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:

G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.

Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.

Your suggestions will be of immense help

Thanks in advance

Re: [ccp4bb] cryo condition

[Tristan Croll]

What about nature's favourite cryoprotectant, trehalose?


From: CCP4 bulletin board  on behalf of Roger Rowlett 

Sent: Tuesday, 5 May 2015 7:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo condition

We rarely use glycerol anymore, because it seems to fail so often for
many of our current proteins. Try glucose, 25-30%. This is most
conveniently done by weighing 125-150 mg of glucose in a microcentrifuge
tube, then addding well solution to the 0.5 mL mark and mixing until
completely dissolved. Then you can try dunking crystals in the cryo
solution, or, you can try the "no-fail" method (which does fail on
occasion) of cryoprotecting directly in the crystallization drop by slow
addition of the cryoprotectant. See
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals.
We have often found the slow addition of a glucose cryoprotectant works
for fragile, high solvent content crystals that are prone to cracking
under osmotic stress.

Other alternatives include high concentrations of sodium malonate
(1-2M), or high concentrations of sodium formate (I think around 4 M?).
These could also be introduced gradually if required.

Good luck!

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 5/4/2015 2:43 PM, Faisal Tarique wrote:
> Hello everyone
>
> Can anybody suggest me a cryo condition for a crystal obtained in
> MIDAS screen of Molecular Dimension:
>
> G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0
>
> G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0
>
> Crystals are in beautiful cuboid shaped but all sorts of PEG
> combinations and Glycerol formulation failed to prevent it from
> cracking and dissolving.
>
> Has anybody faced a similar situation as mentioned above and what
> precaution was taken to prevent it from cracking or dissolving.
>
> Your suggestions will be of immense help
>
> Thanks in advance

Re: [ccp4bb] cryo condition

[Gloria Borgstahl]

High concentration of ammonium formate is a cryosolvent

On Mon, May 4, 2015 at 5:20 PM, Tristan Croll 
wrote:

> What about nature's favourite cryoprotectant, trehalose?
>
> 
> From: CCP4 bulletin board  on behalf of Roger
> Rowlett 
> Sent: Tuesday, 5 May 2015 7:22 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] cryo condition
>
> We rarely use glycerol anymore, because it seems to fail so often for
> many of our current proteins. Try glucose, 25-30%. This is most
> conveniently done by weighing 125-150 mg of glucose in a microcentrifuge
> tube, then addding well solution to the 0.5 mL mark and mixing until
> completely dissolved. Then you can try dunking crystals in the cryo
> solution, or, you can try the "no-fail" method (which does fail on
> occasion) of cryoprotecting directly in the crystallization drop by slow
> addition of the cryoprotectant. See
>
> http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals
> .
> We have often found the slow addition of a glucose cryoprotectant works
> for fragile, high solvent content crystals that are prone to cracking
> under osmotic stress.
>
> Other alternatives include high concentrations of sodium malonate
> (1-2M), or high concentrations of sodium formate (I think around 4 M?).
> These could also be introduced gradually if required.
>
> Good luck!
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>
> On 5/4/2015 2:43 PM, Faisal Tarique wrote:
> > Hello everyone
> >
> > Can anybody suggest me a cryo condition for a crystal obtained in
> > MIDAS screen of Molecular Dimension:
> >
> > G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0
> >
> > G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0
> >
> > Crystals are in beautiful cuboid shaped but all sorts of PEG
> > combinations and Glycerol formulation failed to prevent it from
> > cracking and dissolving.
> >
> > Has anybody faced a similar situation as mentioned above and what
> > precaution was taken to prevent it from cracking or dissolving.
> >
> > Your suggestions will be of immense help
> >
> > Thanks in advance
>

Re: [ccp4bb] Cryo protection

[Faisal Tarique]

Cryo
On 5 May 2015 13:51, faisaltari...@gmail.com wrote:

Thank you everyone..I have got some hints from this discussion and will
definitely try some of them to check its efficacy for my case...Thanx again
for your valuable suggestions..
On 5 May 2015 13:48, "Anthony Savill"  wrote:



Dear colleagues,



Having followed the CCP4 BB discussion on cryoprotection I thought you
might be interested to know that with the help of work from Enrico Stura's
lab Molecular Dimension have introduced two kits now to take some of the
trial and error out of finding an effective cryoprotectant.



http://www.moleculardimensions.com/shopdisplayproducts.asp?id=205&cat=CryoKits



Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.

Crystal Growth & Design,

http://pubs.acs.org/doi/full/10.1021/cg301531f





Tony Savill

Molecular Dimensions Ltd.

Unit 6 Goodwin Business Park

Willie Snaith Road

Newmarket, Suffolk.

UK CB8 7SQ



Tel: +44 1638 561051

Fax: +44 1638 660674



Registered Office

Salisbury House

Station Road

Cambridge

CB1 2LA



Registered in England and Wales:

Registration number 1794026

Re: [ccp4bb] cryo condition

[Clemens Grimm]

Hi Faisal,

Did you try to simply raise the Sokalan CP7 percentage? Additives like  
Glycerol can increase the solubility of proteins, in that case you  
have to counteract by increasing also the precipitant concentration.


If your crystals crack, a likely reason is osmotic shock. Particularly  
large crystals tend to be problematic. It is in general advisable to  
transfer the cryo protectant  onto the crystal within the mother  
liquor rather than fishing the crystal out with a loop. In addition,  
it might be necessary to prepare intermediate concentration of the  
cryo protectant by mixing with reservoir solution and do a stepwise  
increase. I had several projects with very large crystals that  
definitely needed at least five gradual steps over a time span of more  
than half an hour to extract datasets with reasonable mosaicity.


Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight  
sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might  
be worth a try, possibly also in combination with some amount of good  
old glycerol, PEG, Trehalose, TMAO etc.


Best wishes,
Clemens


Zitat von Faisal Tarique :


Hello everyone

Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:

G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.

Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.

Your suggestions will be of immense help

Thanks in advance
--
Regards

Faisal
School of Life Sciences
JNU




--
Dr. Clemens Grimm
Institut für Biochemie
Biozentrum der Universität Würzburg
Am Hubland
D-97074 Würzburg
Germany
e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de
phone : +49 0931 31 84031
-

Re: [ccp4bb] cryo condition

[Matthew Bowler]

Along with all the excellent suggestions so far you can also try no 
cryoprotectant at all: If you harvest your crystal on a mesh loop and 
remove all the mother liquor the crystal lattice itself will act as a 
cryoprotectant as long as the solvent channels are smaller than 40A in 
diameter (they usually are). Shameless self citation: 
http://scripts.iucr.org/cgi-bin/paper?S0907444911031210



Best wishes, Matt.


On 05/05/2015 13:16, Clemens Grimm wrote:

Hi Faisal,

Did you try to simply raise the Sokalan CP7 percentage? Additives like 
Glycerol can increase the solubility of proteins, in that case you 
have to counteract by increasing also the precipitant concentration.


If your crystals crack, a likely reason is osmotic shock. Particularly 
large crystals tend to be problematic. It is in general advisable to 
transfer the cryo protectant  onto the crystal within the mother 
liquor rather than fishing the crystal out with a loop. In addition, 
it might be necessary to prepare intermediate concentration of the 
cryo protectant by mixing with reservoir solution and do a stepwise 
increase. I had several projects with very large crystals that 
definitely needed at least five gradual steps over a time span of more 
than half an hour to extract datasets with reasonable mosaicity.


Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight 
sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might 
be worth a try, possibly also in combination with some amount of good 
old glycerol, PEG, Trehalose, TMAO etc.


Best wishes,
Clemens


Zitat von Faisal Tarique :


Hello everyone

Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:

G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0

G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0

Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.

Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.

Your suggestions will be of immense help

Thanks in advance
--
Regards

Faisal
School of Life Sciences
JNU




--
Dr. Clemens Grimm
Institut für Biochemie
Biozentrum der Universität Würzburg
Am Hubland
D-97074 Würzburg
Germany
e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de
phone : +49 0931 31 84031
-



--
Matthew Bowler
Synchrotron Crystallography Group
European Molecular Biology Laboratory
71 avenue des Martyrs
CS 90181 F-38042 Grenoble
France
===
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.20.71.99

http://www.embl.fr/
===

Re: [ccp4bb] Cryo-EM

[Folmer Fredslund]

Hi Faisal,

I guess a start for you would be to go here:
http://www.ccpem.ac.uk/
and subscribe to that mailing list :-)

Best regards,
Folmer

2015-05-19 9:01 GMT+02:00 Faisal Tarique :

> Hi everyone
>
> A bit off topic..but..I request you to please suggest me some good
> readings related to Cryo-EM..
>
> Thanx in advance
>
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>
>


-- 
Folmer Fredslund

Re: [ccp4bb] Cryo-EM

[Tom Burnley]

Hi Faisal,

* This is a good introduction for a non-microscopist:
Cryo-electron microscopy--a primer for the non-microscopist.
Milne JL1, Borgnia MJ, Bartesaghi A, Tran EE, Earl LA, Schauder DM, Lengyel
J, Pierson J, Patwardhan A, Subramaniam S.
http://www.ncbi.nlm.nih.gov/pubmed/23181775

* A recent review:
How cryo-EM is revolutionizing structural biology
Xiao-chen Bai, Greg McMullan, Sjors H.W Scheres
http://www.sciencedirect.com/science/article/pii/S096800041400187X

* For background theory this is good place to start:
Structural Analysis of Macromolecular Assemblies by Electron Microscopy
E. V. Orlova and H. R. Saibil
http://people.cryst.bbk.ac.uk/~ubcg16z/chemrevsmallfigs.pdf

* And for tools for high-resolution modelling building:
Tools for macromolecular model building and refinement into electron
cryo-microscopy
reconstructions
Alan Brown, Fei Long, Robert A. Nicholls, Jaan Toots, Paul Emsley and Garib
Murshudov
http://www.ncbi.nlm.nih.gov/pubmed/25615868

Folmer beat me to it (thanks!).  Please have a look at http://www.ccpem.ac.
uk/ and subscribe to our mailing list:
https://www.jiscmail.ac.uk/ccpem

All the best,

Tom

On 19 May 2015 at 08:04, Folmer Fredslund  wrote:

> Hi Faisal,
>
> I guess a start for you would be to go here:
> http://www.ccpem.ac.uk/
> and subscribe to that mailing list :-)
>
> Best regards,
> Folmer
>
> 2015-05-19 9:01 GMT+02:00 Faisal Tarique :
>
>> Hi everyone
>>
>> A bit off topic..but..I request you to please suggest me some good
>> readings related to Cryo-EM..
>>
>> Thanx in advance
>>
>> --
>> Regards
>>
>> Faisal
>> School of Life Sciences
>> JNU
>>
>>
>
>
> --
> Folmer Fredslund
>



-- 
Dr Tom Burnley, PhD
CCP-EM
Science and Technology Facilities Council (STFC)
The Research Complex At Harwell
Rutherford Appleton Laboratory, R92
OX11 0FA
01235 56 7871

Re: [ccp4bb] Cryo-EM

[Parthasarathy Sampathkumar]

Hi Faisal,

There are some good video introduction available too:
https://www.youtube.com/watch?v=nkGRhYv01ag

HTH,
Partha

On Tue, May 19, 2015 at 3:01 AM, Faisal Tarique 
wrote:

> Hi everyone
>
> A bit off topic..but..I request you to please suggest me some good
> readings related to Cryo-EM..
>
> Thanx in advance
>
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>
>

Re: [ccp4bb] Cryo-EM

[Mohammad W Bahar]

Hi Faisal,

Two recent reviews well worth reading are below:

A Primer to Single-Particle Cryo-Electron Microscopy.
Cheng Y, Grigorieff N, Penczek PA, Walz T
http://www.ncbi.nlm.nih.gov/pubmed/25910204

Single-Particle Cryo-EM at Crystallographic Resolution.
Cheng Y.
http://www.ncbi.nlm.nih.gov/pubmed/25910205

Best wishes,

Mo


Dr. Mohammad W. Bahar
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
University of Oxford
Roosevelt Drive, Oxford, OX3 7BN
Phone: (+44) 1865 287549
--- Begin Message ---
Hi everyone

A bit off topic..but..I request you to please suggest me some good readings
related to Cryo-EM..

Thanx in advance

-- 
Regards

Faisal
School of Life Sciences
JNU
--- End Message ---

Re: [ccp4bb] Cryo-EM

[Pavel Afonine]

Hi Faisal,

if you are particularly interested in refinement of atomic models into
cryo-EM maps then you may check this too:

http://phenix-online.org/presentations/real_space_refine.pdf

(Of course by no means I claim this being a good reading!)

Good luck,
Pavel


On Tue, May 19, 2015 at 12:01 AM, Faisal Tarique 
wrote:

> Hi everyone
>
> A bit off topic..but..I request you to please suggest me some good
> readings related to Cryo-EM..
>
> Thanx in advance
>
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>
>

Re: [ccp4bb] Cryo-EM

[Yi-Wei Chang]

Hi Faisal,

Here is a new series of lecture video called "Getting Started in 
Cryo-EM" made by Prof. Grant Jensen at Caltech/HHMI.


http://cryo-em-course.caltech.edu/

There are 14.5 hours of lecture total, which starts with the basic 
anatomy of electron microscopes, an introduction to Fourier transforms, 
and the principles of image formation.  Building upon that foundation, 
the lecture then covers sample preparation issues, data collection 
strategies, and basic image processing workflows for tomography, single 
particle analysis, and 2-D crystallography.  It is an excellent 
introduction that will prepare people for real practical training on the 
microscope or to engage in serious conversations about cryo-EM: a great 
way to get started for anyone just joining a cryo-EM lab, or anyone who 
wants to be sure they have the basic concepts down.


Cheers,
Yi-Wei

--
Yi-Wei Chang, Ph.D.
Research Scientist, Jensen lab
California Institute of Technology
1200 E. California Blvd., MC 114-96
Pasadena, CA 91125
Tel:(626)395-8848
Fax:(626)395-5730
ywch...@caltech.edu


On 05/19/2015 12:01 AM, Faisal Tarique wrote:

Hi everyone

A bit off topic..but..I request you to please suggest me some good 
readings related to Cryo-EM..


Thanx in advance

--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] Cryo-EM

[Takanori Nakane]

Hi Faisal,

Recordings of MRC-LMB EM-course last year are available at
http://www2.mrc-lmb.cam.ac.uk/groups/scheres/impact.html

Best regards,

Takanori Nakane

On 2015/05/20 2:36, Yi-Wei Chang wrote:

Hi Faisal,

Here is a new series of lecture video called "Getting Started in
Cryo-EM" made by Prof. Grant Jensen at Caltech/HHMI.

http://cryo-em-course.caltech.edu/

There are 14.5 hours of lecture total, which starts with the basic
anatomy of electron microscopes, an introduction to Fourier transforms,
and the principles of image formation.  Building upon that foundation,
the lecture then covers sample preparation issues, data collection
strategies, and basic image processing workflows for tomography, single
particle analysis, and 2-D crystallography.  It is an excellent
introduction that will prepare people for real practical training on the
microscope or to engage in serious conversations about cryo-EM: a great
way to get started for anyone just joining a cryo-EM lab, or anyone who
wants to be sure they have the basic concepts down.

Cheers,
Yi-Wei

Re: [ccp4bb] Cryo-EM

[Faisal Tarique]

Hi everyone

Thanx a lot..Your suggestions are really informative and a valuable
source of knowledge..

regards

Faisal

On 5/20/15, Takanori Nakane  wrote:
> Hi Faisal,
>
> Recordings of MRC-LMB EM-course last year are available at
> http://www2.mrc-lmb.cam.ac.uk/groups/scheres/impact.html
>
> Best regards,
>
> Takanori Nakane
>
> On 2015/05/20 2:36, Yi-Wei Chang wrote:
>> Hi Faisal,
>>
>> Here is a new series of lecture video called "Getting Started in
>> Cryo-EM" made by Prof. Grant Jensen at Caltech/HHMI.
>>
>> http://cryo-em-course.caltech.edu/
>>
>> There are 14.5 hours of lecture total, which starts with the basic
>> anatomy of electron microscopes, an introduction to Fourier transforms,
>> and the principles of image formation.  Building upon that foundation,
>> the lecture then covers sample preparation issues, data collection
>> strategies, and basic image processing workflows for tomography, single
>> particle analysis, and 2-D crystallography.  It is an excellent
>> introduction that will prepare people for real practical training on the
>> microscope or to engage in serious conversations about cryo-EM: a great
>> way to get started for anyone just joining a cryo-EM lab, or anyone who
>> wants to be sure they have the basic concepts down.
>>
>> Cheers,
>> Yi-Wei
>>
>


-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] cryo protection

[Mark J van Raaij]

you may have thought of this already, but you could try cryoprotection in the 
drop itself.
i.e. slowly adding cryoprotectant to the reservoir, or replacing the reservoir 
bit by bit with solution containing cryoprotectant, and then adding small 
volumes to the side of the drop
- for example, exchanging 20% volume cryo-solution with the reservoir, letting 
equilibrate with the unchanged drop for a few hours, then add 20% volume to the 
drop from the reservoir (i.e. 0.2 ul if the drop is 1 ul), then add another 20% 
of cryo to the reservoir, equilibrate a few hrs, etc. - the idea being to very 
slowly change the drop conditions and minimise risk of cracking.
Of course, you may need patience and many drops of crystals, not just many 
crystals in a few drops, until you find the cryoprotectant where the crystals 
do not crack and still diffract, if you are successful at all...
but if it works, you can just harvest from the equilibrated drop and directly 
flash-cool

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/content/research/macromolecular/mvraaij





On 26 Oct 2011, at 18:46, Leonard Thomas wrote:

> Hi All,
> 
> I have run into a very sensitive crystals system when it comes to cryo 
> protecting them.  I have run through the usual suspects and trays are going 
> to be setup with a cryo protectant as part of crystallization cocktail.  The 
> one problem that  seems to be occurring is that the crystals crack as soon as 
> they are transfered out of the original drop.  I am running out of ideas and 
> really would love some new ones.
> 
> Thanks in advance.
> 
> Len
> 
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
> 
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571

Re: [ccp4bb] cryo protection

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Len,

just to be on the safe side, my list of 'usual suspects' includes
- - glycerol/PEG400
- - LiCl et al at high concentration
- - Butanediol
- - sugars (glucose/ fructose)
- - oil
- - NaMalonate
- - MPD
...

you mention cracking upon transferring the crystal.
- - do you use a pipet for transfer?
- - addition of cryo TO the drop?
- - did you try slow (several minutes - 1hr) / quick addition of cryo
protectant
- - seeding into slightly different conditions/additive screens
- - seeding into cryo conditions
...

How about collecting data at room temperature?

Hope this list contains some new ideas.

Best wishes,
Tim

On 10/26/2011 06:46 PM, Leonard Thomas wrote:
> Hi All,
> 
> I have run into a very sensitive crystals system when it comes to cryo
> protecting them.  I have run through the usual suspects and trays are
> going to be setup with a cryo protectant as part of crystallization
> cocktail.  The one problem that  seems to be occurring is that the
> crystals crack as soon as they are transfered out of the original drop. 
> I am running out of ideas and really would love some new ones.
> 
> Thanks in advance.
> 
> Len
> 
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
> 
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.10 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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GAh002nC1bod6VCBolOv3pQ=
=IO/R
-END PGP SIGNATURE-

Re: [ccp4bb] cryo protection

[Roger Rowlett]

  
  
Len,

We have run into this problem from time to time, and it is very
frustrating. Here are some things to try, some of which you may have
done already:


  Grow crystals in a small percentage of the cryoprotectant
(e.g., 5-10% glycerol). This often allows crystal transfer into
a cryo drop without cracking. (Almost never works for us,
though.)
  Do your crystal transfers in the cold room. This slows
evaporation markedly, and may prevent crystal cracking. (This
works for us some of the time.)
  Transfer your crystals to gradually higher cryoprotectant
concentrations (e.g., to 15% glycerol, then 30% glycerol).
(Fiddly, and the crystals get handled a lot, but often works.)
  
  Use different cryoprotectants. We almost always have fewer
cracking issues with glucose than glycerol, but YMMV.
  
  Avoid transferring the crystal from the drop at all. Just add
cryoprotectant to the drop. Even better, add cryoprotectant to
the drop gradually, while keeping the drop humidified over well
solution. This is our "No-fail" method (this is usually, but not
always successful):

http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals#No_fail_cryoprotection
  
  We typically use glucose in this method, but in principle you
  could try glycerol, MPD, PEG-400, or sodium formate, etc.
  

Otherwise, you can try to grow out of a cryo condition that doesn't
need extra cryoprotectant (been there done that) or give up and
shoot at room temp in-house.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 10/26/2011 12:46 PM, Leonard Thomas wrote:
Hi All,
  
  
  I have run into a very sensitive crystals system when it comes to
  cryo protecting them.  I have run through the usual suspects and
  trays are going to be setup with a cryo protectant as part of
  crystallization cocktail.  The one problem that  seems to be
  occurring is that the crystals crack as soon as they are
  transfered out of the original drop.  I am running out of ideas
  and really would love some new ones.
  
  
  Thanks in advance.
  
  
  Len
  
  
  Leonard Thomas Ph.D.
  
  Macromolecular Crystallography Laboratory Manager
  
  University of Oklahoma
  
  Department of Chemistry and Biochemistry
  
  Stephenson Life Sciences Research Center
  
  101 Stephenson Parkway
  
  Norman, OK 73019-5251
  
  
  lmtho...@ou.edu
  
  http://barlywine.chem.ou.edu
  
  Office: (405)325-1126
  
  Lab: (405)325-7571
  

  

Re: [ccp4bb] cryo protection

[Filip Van Petegem]

Hello Leonard,

one thing to test is whether transferring your crystals to a drop containing
simply well solution also causes cracking. If yes, then the possibility
exists that the absence of protein in solution is causing the trouble. In
that case, you can transfer the crystals to oil:  you'll be transferring the
solution (with protein) in which the crystal grew as well, and slowly remove
it without adding anything 'different'.  However, if your crystals crack
simply because they are mechanically fragile, then the oil may actually be
worse.

Filip

On Wed, Oct 26, 2011 at 9:46 AM, Leonard Thomas  wrote:

> Hi All,
>
> I have run into a very sensitive crystals system when it comes to cryo
> protecting them.  I have run through the usual suspects and trays are going
> to be setup with a cryo protectant as part of crystallization cocktail.  The
> one problem that  seems to be occurring is that the crystals crack as soon
> as they are transfered out of the original drop.  I am running out of ideas
> and really would love some new ones.
>
> Thanks in advance.
>
> Len
>
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
>
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571
>



-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] cryo protection

[Zhou, Tongqing (NIH/VRC) [E]]

You can also try to crosslink before transferring to cryo.


From: Filip Van Petegem 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Wed Oct 26 13:19:16 2011
Subject: Re: [ccp4bb] cryo protection

Hello Leonard,

one thing to test is whether transferring your crystals to a drop containing 
simply well solution also causes cracking. If yes, then the possibility exists 
that the absence of protein in solution is causing the trouble. In that case, 
you can transfer the crystals to oil:  you'll be transferring the solution 
(with protein) in which the crystal grew as well, and slowly remove it without 
adding anything 'different'.  However, if your crystals crack simply because 
they are mechanically fragile, then the oil may actually be worse.

Filip

On Wed, Oct 26, 2011 at 9:46 AM, Leonard Thomas 
mailto:lmtho...@ou.edu>> wrote:
Hi All,

I have run into a very sensitive crystals system when it comes to cryo 
protecting them.  I have run through the usual suspects and trays are going to 
be setup with a cryo protectant as part of crystallization cocktail.  The one 
problem that  seems to be occurring is that the crystals crack as soon as they 
are transfered out of the original drop.  I am running out of ideas and really 
would love some new ones.

Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu<mailto:lmtho...@ou.edu>
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571



--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com<mailto:filip.vanpete...@gmail.com>
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] cryo protection

[Muhammed bashir Khan]

Hi Len;

I was having exactly the same problem with my crystals, but when we grow
the crystals in presence of increasing concentration of Glycerol and MPD
starting from 0.5 to 10%. The crystal doesn't appear after 3% of Glycerol
or MPD but the one which appear in 2.5 to 3 % were much resistant to
cracking than the original crystals.

 Good luck

Bashir

On Wed, October 26, 2011 18:46, Leonard Thomas wrote:
> Hi All,
>
> I have run into a very sensitive crystals system when it comes to cryo
> protecting them.  I have run through the usual suspects and trays are
> going to be setup with a cryo protectant as part of crystallization
> cocktail.  The one problem that  seems to be occurring is that the
> crystals crack as soon as they are transfered out of the original
> drop.  I am running out of ideas and really would love some new ones.
>
> Thanks in advance.
>
> Len
>
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
>
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571
>


-- 
Muhammad Bashir Khan
**
Department for Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Austria

Phone: +43(1)427752224
Fax: +43(1)42779522

Re: [ccp4bb] cryo protection

[Andrew Purkiss-Trew]

Another possibility (other than those already mentioned) is to try  
freezing without a cryoprotectant, by fishing the crystals out onto a  
mesh and removing all the mother liquor.


The following paper has some details:
"Direct cryocooling of naked crystals: are cryoprotection agents  
always necessary?"


Erika Pellegrini, Dario Pianoa, and Matthew W. Bowlera

Acta Cryst. (2011). D67, 902–906

--
Andrew Purkiss
X-ray Laboratory Manager
Cancer Research UK
London Research Institute.


Quoting Leonard Thomas :


Hi All,

I have run into a very sensitive crystals system when it comes to  
cryo protecting them.  I have run through the usual suspects and  
trays are going to be setup with a cryo protectant as part of  
crystallization cocktail.  The one problem that  seems to be  
occurring is that the crystals crack as soon as they are transfered  
out of the original drop.  I am running out of ideas and really  
would love some new ones.


Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571






This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] cryo protection

[Leonard Thomas]

A good number of things to try.  Just a little more info that was  
asked for.  The crystals are grown in Peg 3350 over a range of pH  
values using Bis-Tris Propane.  The are coming out of 2 different salt  
conditions.   My feeling is it is an osmolality problem though I also  
observed cracking when going into a separately made well solution.  I  
will look it trying a number of suggestions given.


Cheers,
Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

On Oct 26, 2011, at 12:54 PM, Muhammed bashir Khan wrote:


Hi Len;

I was having exactly the same problem with my crystals, but when we  
grow
the crystals in presence of increasing concentration of Glycerol and  
MPD
starting from 0.5 to 10%. The crystal doesn't appear after 3% of  
Glycerol

or MPD but the one which appear in 2.5 to 3 % were much resistant to
cracking than the original crystals.

Good luck

Bashir

On Wed, October 26, 2011 18:46, Leonard Thomas wrote:

Hi All,

I have run into a very sensitive crystals system when it comes to  
cryo

protecting them.  I have run through the usual suspects and trays are
going to be setup with a cryo protectant as part of crystallization
cocktail.  The one problem that  seems to be occurring is that the
crystals crack as soon as they are transfered out of the original
drop.  I am running out of ideas and really would love some new ones.

Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571




--
Muhammad Bashir Khan
**
Department for Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Austria

Phone: +43(1)427752224
Fax: +43(1)42779522

Re: [ccp4bb] cryo protection

[harkewal singh]

Len,
May be you have already done this. I would closely check my
crystallization conditions and also check the pH of the cryo. In some
cases, during cryoprotection the pH of the original drop may drastically
different than the cryo solution. Also, sometime back, we were exploring
different cryoprotectant conditions for sensitive crystal and came
across this -
http://www.xtals.org/crystal_cryo.pdf by Artem. 

HTH
Harkewal


On Wed, 26 Oct 2011 11:46:08 -0500, Leonard Thomas 
wrote:
> Hi All,
> 
> I have run into a very sensitive crystals system when it comes to
> cryo  protecting them.  I have run through the usual suspects and
> trays are  going to be setup with a cryo protectant as part of
> crystallization  cocktail.  The one problem that  seems to be
> occurring is that the  crystals crack as soon as they are transfered
> out of the original  drop.  I am running out of ideas and really would
> love some new ones.
> 
> Thanks in advance.
> 
> Len
> 
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
> 
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571

Re: [ccp4bb] cryo protection

[Mathews, Irimpan I.]

One small point:

Just make sure that you are not too off from the contents of the protein 
solution.  Sometimes protein solution may have a high amount of salt or things 
like that and we forget to include atleast half of this concentration into the 
cryo solution.  This could easily crack the crystals depending on the 
concentration and the type of compounds in it.

Regards,
Mathews


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Leonard 
Thomas
Sent: Wednesday, October 26, 2011 11:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo protection

A good number of things to try.  Just a little more info that was  
asked for.  The crystals are grown in Peg 3350 over a range of pH  
values using Bis-Tris Propane.  The are coming out of 2 different salt  
conditions.   My feeling is it is an osmolality problem though I also  
observed cracking when going into a separately made well solution.  I  
will look it trying a number of suggestions given.

Cheers,
Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

On Oct 26, 2011, at 12:54 PM, Muhammed bashir Khan wrote:

> Hi Len;
>
> I was having exactly the same problem with my crystals, but when we  
> grow
> the crystals in presence of increasing concentration of Glycerol and  
> MPD
> starting from 0.5 to 10%. The crystal doesn't appear after 3% of  
> Glycerol
> or MPD but the one which appear in 2.5 to 3 % were much resistant to
> cracking than the original crystals.
>
> Good luck
>
> Bashir
>
> On Wed, October 26, 2011 18:46, Leonard Thomas wrote:
>> Hi All,
>>
>> I have run into a very sensitive crystals system when it comes to  
>> cryo
>> protecting them.  I have run through the usual suspects and trays are
>> going to be setup with a cryo protectant as part of crystallization
>> cocktail.  The one problem that  seems to be occurring is that the
>> crystals crack as soon as they are transfered out of the original
>> drop.  I am running out of ideas and really would love some new ones.
>>
>> Thanks in advance.
>>
>> Len
>>
>> Leonard Thomas Ph.D.
>> Macromolecular Crystallography Laboratory Manager
>> University of Oklahoma
>> Department of Chemistry and Biochemistry
>> Stephenson Life Sciences Research Center
>> 101 Stephenson Parkway
>> Norman, OK 73019-5251
>>
>> lmtho...@ou.edu
>> http://barlywine.chem.ou.edu
>> Office: (405)325-1126
>> Lab: (405)325-7571
>>
>
>
> -- 
> Muhammad Bashir Khan
> **
> Department for Structural and Computational Biology
> Max F. Perutz Laboratories
> University of Vienna
> Campus Vienna Biocenter 5
> A-1030 Vienna
> Austria
>
> Austria
>
> Phone: +43(1)427752224
> Fax: +43(1)42779522
>
>

Re: [ccp4bb] cryo protection

[David Schuller]

One more thing you could try: high pressure cryo-cooling. Se any of a 
number of paperas by Chae Un Kim; e.g.


http://www.ncbi.nlm.nih.gov/pubmed/17452791

Acta Crystallogr D Biol Crystallogr. 
 2007 May;63(Pt 5):653-9. 
Epub 2007 Apr 21.





On 10/26/11 12:46, Leonard Thomas wrote:

Hi All,

I have run into a very sensitive crystals system when it comes to cryo 
protecting them.  I have run through the usual suspects and trays are 
going to be setup with a cryo protectant as part of crystallization 
cocktail.  The one problem that  seems to be occurring is that the 
crystals crack as soon as they are transfered out of the original 
drop.  I am running out of ideas and really would love some new ones.


Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571



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

Re: [ccp4bb] cryo protection

[Jim Pflugrath]

For some ideas on cryocrystallography, one can watch an online webinar on the 
subject:
http://www.rigaku.com/protein/webinar-001.html

Maybe some "unbiased" folks can comment? ;)

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Leonard Thomas 
[lmtho...@ou.edu]
Sent: Wednesday, October 26, 2011 11:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo protection

Hi All,

I have run into a very sensitive crystals system when it comes to cryo
protecting them.  I have run through the usual suspects and trays are
...

Re: [ccp4bb] cryo protection

[Jens Kaiser]

Hey Len,
  I had this problem, too. As you know, my favorite first try is always
fomblin (no need to mix anything). I had quite a bit success in stubborn
cases to inject about 4uL fomblin through the tape on top of the drop
and then looping crystals through the oil layer. You can wick the mother
liquor off and try them right away or continue manipulation under oil

Cheers,

Jens

 On Wed, 2011-10-26 at 11:46 -0500, Leonard Thomas wrote:
> Hi All,
> 
> I have run into a very sensitive crystals system when it comes to cryo  
> protecting them.  I have run through the usual suspects and trays are  
> going to be setup with a cryo protectant as part of crystallization  
> cocktail.  The one problem that  seems to be occurring is that the  
> crystals crack as soon as they are transfered out of the original  
> drop.  I am running out of ideas and really would love some new ones.
> 
> Thanks in advance.
> 
> Len
> 
> Leonard Thomas Ph.D.
> Macromolecular Crystallography Laboratory Manager
> University of Oklahoma
> Department of Chemistry and Biochemistry
> Stephenson Life Sciences Research Center
> 101 Stephenson Parkway
> Norman, OK 73019-5251
> 
> lmtho...@ou.edu
> http://barlywine.chem.ou.edu
> Office: (405)325-1126
> Lab: (405)325-7571

Re: [ccp4bb] cryo protection

[James Holton]

I have always been a fan of oil, which has already been suggested.  Have 
you tried that?


Cross-linking has already been suggested, and these are some good protocols:
Lusty (1999) J. Appl. Crystallogr. 32, 106-112.
McWhirter, et al. (1999) PNAS USA 96, 8408-8413.

In the latter paper the crystals cracked immediately upon breaking the 
seal on the cover slip (limbo trays).  The cross linker was introduced 
with a Hamilton syringe via a pre-cut and grease-filled hole in the 
crystallization chamber.  That way there were no mechanical vibrations 
at all.  You do NOT need to add it directly to the drop.  Gluteraldehyde 
has sufficient vapor pressure to permeate slowly into it.  After a few 
days, the crystals were incredibly robust, and gave the best 
diffraction.  The only problem with gluteraldehyde is if you have 
primary amines in your buffer, such as tris.  If that is the case, you 
can usually substitute bis-tris, or just use a different kind of 
crosslinker.


Another trick I like if you have a "cryo" component in the mother liquor 
(protein counts) is to just let the drop dry up slowly.  You can keep 
"sampling" it with a small loop (removes ~1 nl) until you see it 
flash-cool clear.  Then, if the crystals survived, you can flash-cool 
them in the dried-down mother liquor.  This worked for me once with 
crystals that just didn't want to transfer into anything.


-James Holton
MAD Scientist

On 10/26/2011 9:46 AM, Leonard Thomas wrote:

Hi All,

I have run into a very sensitive crystals system when it comes to cryo 
protecting them.  I have run through the usual suspects and trays are 
going to be setup with a cryo protectant as part of crystallization 
cocktail.  The one problem that  seems to be occurring is that the 
crystals crack as soon as they are transfered out of the original 
drop.  I am running out of ideas and really would love some new ones.


Thanks in advance.

Len

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

Re: [ccp4bb] cryo conditions

[Ulrike Demmer]

Dear Ed,

try ca. 30 % PEG 400
Another possibility could be Perfluoropolyether Cryo Oil from Hampton Research. 
This liquid doesn't mix with water. You put the crystal in few microliter and 
remove the excess water droplets from your screen solution (otherwise you will 
get ice rings).

Cheers,

Ulrike

Re: [ccp4bb] cryo conditions

[Enrico Stura]

Dear Ed,

Glycerol is a solubilizing agent. Unless something is done to conteract  
such effect
the crystals will dissolve. Laura Vera from my lab made a presentation at  
ICCBM-14

on this subject.
We have developed a set of balanced solutions, mixed solubilizers like  
glycerol and ethylene
glycol with precipitants like MPD, DMSO and neutral compounds like  
di-ethylene glycol
and propanediol that with PEG and salts crystallization conditions take  
out the guesswork

from designing cryo-conditions.
The mixed conpounds are marketed by Molecular Dimensions
google search: CryoProtX

Before ordering the kit check that the crystals do not dissolve with MPD.  
If crystals crack
but do not dissolve with MPD (it may be worth ordering the kit). Cracking  
is good since it means
that the crystals do indeed dissolve in glycerol because of glycerol's  
solubilizing properties.


Now try Propylene glycol (1,2-propanediol).
This is a neutral single component. It is a good starting point to work on  
your cryo-conditions
which you will then be able to improve when you have all the various  
cryo-components and

premixed combinations.

The paper for the mixed cryosolutions will be submitted to the ICCBM-14  
special issue

of the Crystal Growth and Design probably this week.

polyacrylic acid is a novell precipitant, make sure that you keep  
magnesium in your
cryosolution. I would also check if increasing the magnesium concentration  
in the

cryoprotectant solution might not be beneficial.

Enrico.

On Wed, 17 Oct 2012 06:01:08 +0200, Edward A. Berry   
wrote:



Would someone suggest a cryoprotectant for index screen #59? It contains
0.02 M MgCl2
0.1 M HEPES pH 7.5
22% polyacrylic acid 5100, Na salt

Adding glycerol tends to dissolve the crystals.

Thanks,
Ed



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryo conditions

[Edward A. Berry]

Thanks to all for the suggestions! We will try, this weekend at macCHESS,
all those conditions for which we have reagents, and order the others now.

> On Oct 16, 2012, at 9:01 PM, "Edward A. Berry"  wrote:
>
>> Would someone suggest a cryoprotectant for index screen #59? It contains
>> 0.02 M MgCl2
>> 0.1 M HEPES pH 7.5
>> 22% polyacrylic acid 5100, Na salt
>>
>> Adding glycerol tends to dissolve the crystals.
>>
>> Thanks,
>> Ed
>


Yvonne TAN Yih Wan (ETC) wrote:

I had a similar condition. I have used increased concentration of polyacrylic 
acid 5100 as cryo. Gradual increase of  10%glycerol + 10% E.glycol in mother 
liqour can aslo be used as cryo, in my case.


Roger Rowlett wrote:

Glucose, 15-25% depending on how good polyacrylic acid is as a cryoptectant 
itself, is worth a try. It doesn't
usually dissolve proteins as readily as glycerol. Ethylene glycol is also an 
option.



borland...@gmail.com wrote:

I obtain crystals in the same condition. Obviously the cryo that works for me 
may not work for other proteins, but I use 10% ethylene glycol and soak the 
crystals  anywhere from 10 minutes to one hour. The other thing that seems to 
make a huge difference for me is the way in which I cool the crystals. 
Sometimes freezing in liquid nitrogen versus freezing directly in the 
cryostream can be the difference between 5 angstrom high mosaicicity data and 2 
angstrom high quality data.  I have obtained good data from both freezing 
processes.
Hope this helps, it would be interesting to know how it turns out for you.

*note: the above methods are for a membrane protein. May or may not make a 
difference for soluble proteins.



Francis E Reyes wrote:

Oil?



David Cobessi wrote:

Dear Ed,
Have you tried paratone or another oil?
Best regards,
David


Benz, Joerg wrote:


did you try paraffin oil already (dry oil,  can be found i.e. in the Cryokit 
from Hampton). Worked for me in nearly all difficult cases. You just need to 
make sure that all of the surrounding solution around the crystal is replaced 
by the oil.



Ulrike Demmer wrote:


try ca. 30 % PEG 400
Another possibility could be Perfluoropolyether Cryo Oil from Hampton Research. 
This liquid doesn't mix with water. You put the crystal in few microliter and 
remove the excess water droplets from your screen solution (otherwise you will 
get ice rings).



Anthony Savill wrote:


Please have a look at our new product CryoProtX.

http://www.moleculardimensions.com/shopexd.asp?id=3732



Enrico Stura wrote:

Glycerol is a solubilizing agent. Unless something is done to conteract such 
effect
the crystals will dissolve. Laura Vera from my lab made a presentation at
ICCBM-14 on this subject.
We have developed a set of balanced solutions, mixed solubilizers like glycerol 
and ethylene
glycol with precipitants like MPD, DMSO and neutral compounds like di-ethylene 
glycol
and propanediol that with PEG and salts crystallization conditions take
out the guesswork from designing cryo-conditions.
The mixed conpounds are marketed by Molecular Dimensions google search: 
CryoProtX

Before ordering the kit check that the crystals do not dissolve with MPD. If 
crystals crack
but do not dissolve with MPD (it may be worth ordering the kit). Cracking is 
good since it means
that the crystals do indeed dissolve in glycerol because of glycerol's
solubilizing properties.

Now try Propylene glycol (1,2-propanediol).
This is a neutral single component. It is a good starting point to work on
your cryo-conditions which you will then be able to improve when you have all 
the various
cryo-components and premixed combinations.

The paper for the mixed cryosolutions will be submitted to the ICCBM-14
special issue of the Crystal Growth and Design probably this week.

polyacrylic acid is a novell precipitant, make sure that you keep
magnesium in your cryosolution. I would also check if increasing the magnesium 
concentration
in the cryoprotectant solution might not be beneficial.




On Oct 16, 2012, at 9:01 PM, "Edward A. Berry"  wrote:


Would someone suggest a cryoprotectant for index screen #59? It contains
0.02 M MgCl2
0.1 M HEPES pH 7.5
22% polyacrylic acid 5100, Na salt

Adding glycerol tends to dissolve the crystals.

Thanks,
Ed

Re: [ccp4bb] cryo condition

[Enrico Stura]

80% saturated Li2SO4


On Thu, 23 May 2013 11:42:09 +0200, Faisal Tarique  
 wrote:



Dear all

Can anybody tell me the appropriate cryo condition for the crystals
obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10%
glycerol to it but still the ice ring is forming..

Thanx in advance



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] cryo condition

[Boaz Shaanan]

Hi,


Paraton oil worked nicely for me for these conditions.


          Boaz 
 
Boaz Shaanan, Ph.D.

Dept. of Life Sciences  
Ben-Gurion University of the Negev  
Beer-Sheva 84105    
Israel  
    
E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan  
Fax:   972-8-647-2992 or 972-8-646-1710
 
 








From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique [faisaltari...@gmail.com]
Sent: Thursday, May 23, 2013 12:42 PM
To: 
CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo condition




Dear all


Can anybody tell me the appropriate cryo condition for the crystals obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but still the ice ring is forming..


Thanx in advance
-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] cryo condition

[Roger Rowlett]

30% glycerol or 25% glucose should be sufficient for 1-2 M ammonium sulfate.

Roger Rowlett
On May 23, 2013 5:42 AM, "Faisal Tarique"  wrote:

>
> Dear all
>
> Can anybody tell me the appropriate cryo condition for the crystals
> obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10%
> glycerol to it but still the ice ring is forming..
>
> Thanx in advance
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU
>

Re: [ccp4bb] cryo condition

[Janet Newman]

I would try 2.5M or 3M sodium malonate pH 7

Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: 23 May 2013 21:21
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo condition


30% glycerol or 25% glucose should be sufficient for 1-2 M ammonium sulfate.

Roger Rowlett

On May 23, 2013 5:42 AM, "Faisal Tarique" 
mailto:faisaltari...@gmail.com>> wrote:

Dear all

Can anybody tell me the appropriate cryo condition for the crystals obtained in 
2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but 
still the ice ring is forming..

Thanx in advance
--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] cryo condition

[jan]

Hi Faisal,
3 - 3.5M Ammonium sulphate with your buffer should work too.
Take a small loop.

Jan
--
Jan Abendroth
Emerald BioStructures
Seattle / Bainbridge Island WA, USA
home: Jan.Abendroth_at_gmail.com
work: JAbendroth_at_embios.com
http://www.emeraldbiostructures.com

On May 23, 2013, at 2:42 AM, Faisal Tarique  wrote:

> 
> Dear all
> 
> Can anybody tell me the appropriate cryo condition for the crystals obtained 
> in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but 
> still the ice ring is forming..
> 
> Thanx in advance
> -- 
> Regards
> 
> Faisal
> School of Life Sciences
> JNU

Re: [ccp4bb] cryo condition

[Matthew BOWLER]

Hi Faisal,


On 2013-05-23 11:42, Faisal Tarique wrote:

Dear all

Can anybody tell me the appropriate cryo condition for the crystals
obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10%
glycerol to it but still the ice ring is forming..

Thanx in advance--
Regards

Faisal
School of Life Sciences
JNU


--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
===
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04

http://www.embl.fr/
===

Re: [ccp4bb] cryo condition

[Matthew BOWLER]

Hi Faisal,
if your solvent channels are smaller than 40A in the largest dimension 
(most are) you can use a mesh loop to pick up the crystal and then wick 
away all of the mother liquor. You can then flash cool your crystal 
without having to transfer the crystal to another solution. Good luck, 
Matt




On 2013-05-23 11:42, Faisal Tarique wrote:

Dear all

Can anybody tell me the appropriate cryo condition for the crystals
obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10%
glycerol to it but still the ice ring is forming..

Thanx in advance--
Regards

Faisal
School of Life Sciences
JNU


--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
===
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04

http://www.embl.fr/
===

Re: [ccp4bb] cryo condition

[Edward Snell]

Just to follow up, the paper that was attached is based on the far more 
original work by Garman and Mitchell

Elspeth Garman and Edward Mitchell. (1996) Glycerol concentrations required for 
cryoprotection of 50 typical protein crystallisation solutions. J.Appl. Cryst. 
29, 584-587.

And there is also a further paper

Glycerol concentrations required for the successful vitrification of cocktail 
conditions in a high-throughput crystallization screen.Kempkes R, Stofko E, Lam 
K, Snell EH.(2003) Acta Cryst D 64, 287-301.

There are a few other papers on high salt concentrations as a cryoprotectant 
including:

Todd Holyoak, Timothy D. Fenn, Mark A. Wilson, Aaron G. Moulin, Dagmar Ringe 
and Gregory A. Petsko. (2003) Malonate: a versatile cryoprotectant and 
stabilizing solution for salt-grown macromolecular crystals. Acta D. 59, 
2356-2358.

Cheers,

Eddie.

Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Senior Scientist, Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660 
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu  
Telepathy: 42.2 GHz

Heisenberg was probably here!


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thibaut 
Crepin
Sent: Thursday, May 23, 2013 6:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo condition

Dear Faisal,

this paper can be really useful.

Regards
Thibaut


On 23/05/2013 11:42, Faisal Tarique wrote:
>
> Dear all
>
> Can anybody tell me the appropriate cryo condition for the crystals 
> obtained in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% 
> glycerol to it but still the ice ring is forming..
>
> Thanx in advance
> --
> Regards
>
> Faisal
> School of Life Sciences
> JNU

Re: [ccp4bb] cryo condition

[Tanner, John J.]

L-proline works well with ammonium sulfate:

http://www.ncbi.nlm.nih.gov/pubmed/22868767


Sent from Jack's iPad

On May 23, 2013, at 4:42 AM, "Faisal Tarique"  wrote:

> 
> Dear all
> 
> Can anybody tell me the appropriate cryo condition for the crystals obtained 
> in 2M Ammonium sulphate and tris pH8.5..I tried to add 10% glycerol to it but 
> still the ice ring is forming..
> 
> Thanx in advance
> -- 
> Regards
> 
> Faisal
> School of Life Sciences
> JNU

Re: [ccp4bb] cryo condition

[Ed Pozharski]

Matt,

with this technique, how do you prevent crystal from drying up (other
than "doing it fast")?  I know Thorne's group does this trick under oil.
If you take no extra precautions, do you have an estimate of how often
diffraction is destroyed by this?  

On the other hand, it's quite possible that what destroys resolution
when crystals dry up is increase in concentration of non-volatile mother
liquor components, which shouldn't be happening here to the same degree.

Cheers,

Ed.

On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote:
> Hi Faisal,
> if your solvent channels are smaller than 40A in the largest dimension 
> (most are) you can use a mesh loop to pick up the crystal and then wick 
> away all of the mother liquor. You can then flash cool your crystal 
> without having to transfer the crystal to another solution. Good luck, 
> Matt

-- 
After much deep and profound brain things inside my head, 
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] cryo condition

[Matthew BOWLER]

Hi Ed,
good question. I have found that you have a good 30 seconds to remove 
the surrounding liquid - so while you have to do it fast you have enough 
time that it doesn't need a robot and even a malcoordinate such as 
myself can do it. I'm afraid that I have no estimate for how often 
diffraction is lost this way  more info here 
http://scripts.iucr.org/cgi-bin/paper?S0907444911031210


I started looking at this when doing dehydration experiments where it 
has long been observed that you can directly cool the crystals after 
dehydration - I have a feeling that 'over drying' a crystal leads to a 
complete loss of lattice order - I have no evidence for this but all 
systems that we have looked at that benefit from dehydration, or not, 
come to quite a sharp cutoff where suddenly there is no more 
diffraction, one can often recover diffraction by rehydration.  This 
could of course be due to too high a concentration of mother liquor but 
quite often it occurs at relative humidity values  Cheers, Matt.







On 2013-05-23 15:32, Ed Pozharski wrote:

Matt,

with this technique, how do you prevent crystal from drying up (other
than "doing it fast")?  I know Thorne's group does this trick under 
oil.
If you take no extra precautions, do you have an estimate of how 
often

diffraction is destroyed by this?

On the other hand, it's quite possible that what destroys resolution
when crystals dry up is increase in concentration of non-volatile 
mother
liquor components, which shouldn't be happening here to the same 
degree.


Cheers,

Ed.

On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote:

Hi Faisal,
if your solvent channels are smaller than 40A in the largest 
dimension
(most are) you can use a mesh loop to pick up the crystal and then 
wick

away all of the mother liquor. You can then flash cool your crystal
without having to transfer the crystal to another solution. Good 
luck,

Matt


--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
===
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04

http://www.embl.fr/
===

Re: [ccp4bb] cryo condition

[Matthew BOWLER]

I keep sending mails by accident today - apologies for the spam.  The 
last sentence of my should read:


This could of course be due to too high a concentration of mother 
liquor but quite often it occurs at relative humidity values where the 
concentration of the mother liquor components will not have increased by 
very much. Cheers, Matt.


On 2013-05-23 15:32, Ed Pozharski wrote:

Matt,

with this technique, how do you prevent crystal from drying up (other
than "doing it fast")?  I know Thorne's group does this trick under 
oil.
If you take no extra precautions, do you have an estimate of how 
often

diffraction is destroyed by this?

On the other hand, it's quite possible that what destroys resolution
when crystals dry up is increase in concentration of non-volatile 
mother
liquor components, which shouldn't be happening here to the same 
degree.


Cheers,

Ed.

On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote:

Hi Faisal,
if your solvent channels are smaller than 40A in the largest 
dimension
(most are) you can use a mesh loop to pick up the crystal and then 
wick

away all of the mother liquor. You can then flash cool your crystal
without having to transfer the crystal to another solution. Good 
luck,

Matt


--
Matthew Bowler
Synchrotron Science Group
European Molecular Biology Laboratory
BP 181, 6 rue Jules Horowitz
38042 Grenoble Cedex 9
France
===
Tel: +33 (0) 4.76.20.76.37
Fax: +33 (0) 4.76.88.29.04

http://www.embl.fr/
===

Re: [ccp4bb] cryo condition

[Faisal Tarique]

Thank you everybody for their nice suggestions..

On Thu, May 23, 2013 at 7:39 PM, Matthew BOWLER  wrote:

> I keep sending mails by accident today - apologies for the spam.  The last
> sentence of my should read:
>
> This could of course be due to too high a concentration of mother liquor
> but quite often it occurs at relative humidity values where the
> concentration of the mother liquor components will not have increased by
> very much. Cheers, Matt.
>
>
> On 2013-05-23 15:32, Ed Pozharski wrote:
>
>> Matt,
>>
>> with this technique, how do you prevent crystal from drying up (other
>> than "doing it fast")?  I know Thorne's group does this trick under oil.
>> If you take no extra precautions, do you have an estimate of how often
>> diffraction is destroyed by this?
>>
>> On the other hand, it's quite possible that what destroys resolution
>> when crystals dry up is increase in concentration of non-volatile mother
>> liquor components, which shouldn't be happening here to the same degree.
>>
>> Cheers,
>>
>> Ed.
>>
>> On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote:
>>
>>> Hi Faisal,
>>> if your solvent channels are smaller than 40A in the largest dimension
>>> (most are) you can use a mesh loop to pick up the crystal and then wick
>>> away all of the mother liquor. You can then flash cool your crystal
>>> without having to transfer the crystal to another solution. Good luck,
>>> Matt
>>>
>>
> --
> Matthew Bowler
> Synchrotron Science Group
> European Molecular Biology Laboratory
> BP 181, 6 rue Jules Horowitz
> 38042 Grenoble Cedex 9
> France
> ==**=
> Tel: +33 (0) 4.76.20.76.37
> Fax: +33 (0) 4.76.88.29.04
>
> http://www.embl.fr/
> ==**=
>



-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] cryo-EM

[Rob Nicholls]

Dear Xavier,

I recommend having a look at a few of the articles that were published 
in the special issue of Acta Cryst D from June this year - Proceedings 
of the third CCP-EM Spring Symposium.


https://journals.iucr.org/d/issues/2018/06/00/

Best regards,
Rob



-- Original Message --
From: "F.Xavier Gomis-Rüth" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: 31/08/2018 12:52:48
Subject: [ccp4bb] cryo-EM


Dear all,

is there some kind of general trend if not consensus as how to refine 
cryo-EM structures? What do people do for maps in the 3-4Å range,


pure RSR against the untouched map and that's it? Reciprocal space 
refinement against the backtransformed structure factors from the map


but without touching the map?

I've even heard that some people "dare" to calculate 2mFo-DFc-maps

Thanks a lot for your feedback.

Best,

Xavier

--



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Re: [ccp4bb] cryo-EM

[Bruno KLAHOLZ]

Dear Xavier, dear all,

good points indeed. I guess the aim is to refine an atomic model against a 
cryo-EM map so that the atomic model describes the experimental data well, 
aiming for a good final geometry, refined B-factors etc. of the atomic model.
Here is a description of what we recently did for one of our structures: 
https://www.nature.com/protocolexchange/protocols/6365

HTH,

Bruno



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Israel 
Sanchez
Sent: Friday, August 31, 2018 2:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo-EM

Dear Xavier,

On top of Rob's excellent suggestion, I would recoment you to have a look at 
this paper by Alan Brown and co-workers (including Rob himself):

https://www.ncbi.nlm.nih.gov/pubmed/25615868

We normally performs an initial real-space refinement with phenix.real-space 
followed by two step of reciprocal space refinement with Refmac. In the first 
Refmac run we normally use secondary structure restrains generated by ProSmart 
for proteins and LibG for nuclei acids. In the second Refmac run, we try to 
release the restrains power and also play around with the matrix governing the 
weight between the experimental data and ideal symmetry. You can monitor when 
are you entering over-fitting  conditions employing the half maps normally 
output by Relion (or the program you use for cryoEM data processing). More 
specific examples here:

https://www.ncbi.nlm.nih.gov/pubmed/24675956

and here:

https://www.ncbi.nlm.nih.gov/pubmed/24792965

Finally, I would not recommend at all playing around with the phases of a 
cryoEM map. If you have a region which is less visible due to poorer local 
resolution, you may blur the map computing a post-precessed map with a lower 
Bfactor that the one automatically assigned by Relion or low pass filter the 
same map a bit. Or even combine both approaches. Not sure what the big guys in 
the field think about tinkering with the phases of a cryoEM map but I feel this 
is still highly controversial. May be Garib or Pavel think differently?

Hope this help.



El vie., 31 ago. 2018 a las 8:02, Rob Nicholls 
(mailto:nicho...@mrc-lmb.cam.ac.uk>>) escribió:
Dear Xavier,

I recommend having a look at a few of the articles that were published in the 
special issue of Acta Cryst D from June this year - Proceedings of the third 
CCP-EM Spring Symposium.

https://journals.iucr.org/d/issues/2018/06/00/

Best regards,
Rob



-- Original Message --
From: "F.Xavier Gomis-Rüth" mailto:xgr...@ibmb.csic.es>>
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Sent: 31/08/2018 12:52:48
Subject: [ccp4bb] cryo-EM


Dear all,

is there some kind of general trend if not consensus as how to refine cryo-EM 
structures? What do people do for maps in the 3-4Å range,

pure RSR against the untouched map and that's it? Reciprocal space refinement 
against the backtransformed structure factors from the map

but without touching the map?

I've even heard that some people "dare" to calculate 2mFo-DFc-maps

Thanks a lot for your feedback.

Best,

Xavier
--
[cid:part1.305680E4.1605AAD1@ibmb.csic.es]



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--
Israel S. Fernandez PhD
Ramakrishnan-Lab
MRC Laboratory of Molecular Biology,
Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.



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Re: [ccp4bb] cryo-EM

[Pavel Afonine]

Hi Xavier,

> is there some kind of general trend if not consensus as how to refine
> cryo-EM structures?
>
Not really consensus, but my clearly biased contribution to the topic:

I'd say basic common sense for refinement applies:

- optimally sharpen the map (
https://journals.iucr.org/d/issues/2018/06/00/ic5102/);

- refine against the map using refinement parameterization appropriate for
the data (map) quality (resolution) (
https://journals.iucr.org/d/issues/2018/06/00/ic5103/);

- make sure to validate data (map), model, and model-to-map fit (
https://doi.org/10.1101/279844).

> What do people do for maps in the 3-4Å range,
>
3-4A may require secondary structure and rotamer restraints. Use "NCS" if
available. If map is symmetrized, then use "NCS" constraints, otherwise
restraints should be just fine. Typically, data at this resolution range
does not allow to confirm geometry validation outliers, so your aim is no
outleirs. Etc, etc. And again, validate the model and model-to-map fit,
locally (at atom/residue level) and globally (standard "Table 1" metrics,
discussed here https://doi.org/10.1101/279844 , for example).

> pure RSR against the untouched map and that's it?
>
Good idea, in my opinion.

> Reciprocal space refinement against the backtransformed structure factors
> from the map but without touching the map?
>
Personally don't like this idea.

> I've even heard that some people "dare" to calculate 2mFo-DFc-maps
>
Not a good idea, I'm pretty sure. Cryo-EM map is not biased by atomic model
(normally, unless a model was used in reconstruction). 2mFo-DFc map is
model biased by definition. Also needless to say that absence of "Fobs" in
cryo-EM is a good hint that this map isn't what you want -;)

All the best,
Pavel



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Re: [ccp4bb] Cryo tongs/manipulator

[James Holton]

These just look like ordinary hemostats that have been dipped in rubber 
goo stuff.  You can get rubber goo stuff from McMaster-Carr:

http://www.mcmaster.com/#plastic-coating-dip/=7l6uk4

-James Holton
MAD Scientist

On 6/18/2010 10:12 AM, Bandaranayake, Rajintha wrote:

Hello all,
Does anyone know where one could purchase cryo-vial tongs/manipulatos 
with the plastic coated handles (See attached image)? We have checked 
with Blake Industries (where I think we originally got them eons ago), 
however their new catalog does not list them any more.


Thanks in advance.
-Rajintha 

Re: [ccp4bb] Cryo tongs/manipulator

[William G. Scott]

When searching, avoid the almost overwhelming temptation to spell it as
"vile manipulator" or you will just wind up with a listing of university
administrators.



Bandaranayake, Rajintha wrote:
> Hello all,
> Does anyone know where one could purchase cryo-vial tongs/manipulatos with
> the plastic coated handles (See attached image)? We have checked with
> Blake Industries (where I think we originally got them eons ago), however
> their new catalog does not list them any more.
>
> Thanks in advance.
> -Rajintha
>

Re: [ccp4bb] Cryo with succinate

[Van Den Berg, Bert]

It should be pretty straightforward to figure that out by freezing and shooting 
loops with mother liquor only. You can easily fine-tune by including any 
additional buffer components you may have.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Tjaard Pijning
Sent: Tue 7/22/2008 8:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryo with succinate


I have crystals grown from 1 M sodium succinate pH 7 and have been searching 
for suitable cryo conditions.
Is succinate in itself already a cryoprotectant (maybe at somewhat higher 
concentration) or should I
add e.g. glycerol ? Any experience or ideas are welcome.
 
Thanks in advance,
  

 

Ing. Tjaard Pijning

Protein Crystallography Group
University of Groningen
Nijenborgh 4
9747 AG Groningen
The Netherlands
Tel. (31)(0)50 363 4385

 

<><>

Re: [ccp4bb] Cryo with succinate

[Roger Rowlett]

Tjaard Pijning wrote:
I have crystals grown from 1 M sodium succinate pH 7 and have 
been searching for suitable cryo conditions.
Is succinate in itself already a cryoprotectant (maybe at somewhat 
higher concentration) or should I

add e.g. glycerol ? Any experience or ideas are welcome.
 
Thanks in advance,
 


***   **Ing. Tjaard Pijning*

Protein Crystallography Group
University of Groningen
Nijenborgh 4
9747 AG Groningen
The Netherlands
Tel. (31)(0)50 363 4385

 

Adding 25-30% glucose is sufficient to cryoprotect almost any 
crystallization solution from ice formation. If your crystals can't 
tolerate transfer to M.L. + glucose without cracking, you can try adding 
the cryoprotectant solution gradually to the drop the crystals are in. I 
usually add M.L. + cryoprotectant at 125% of the final concentration in 
0.25, 0.25, 0.5, 1.0, and 2.0 drop volumes a few minutes apart to allow 
for mixing and equilibration. (You can do this over the original well to 
avoid excessive evaporation.)


Alternatively, you can freeze empty drops with various amounts of 
glycerol and examine the frozen loops. If your solutions freeze clear, 
you probably have enough glycerol to prevent ice formation.


Cheers,


--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]

Re: [ccp4bb] Cryo with succinate

[Alastair McEwen]

I have recently been working with crystals grown 
from 1M sodium succinate at pH 7.5 and I have 
found that 20% ethylene glycol worked well as a 
cryoprotectant. At concentrations below this we 
had ice formation. We did not do an exhaustive 
search for a cryo but found that we had very poor 
diffraction with glycerol, parrafin oil and various Fomblin oils.


Hope this helps,

Alastair


Le 14:41 22/07/2008,Tjaard Pijning écrit:
I have crystals grown from 1 M sodium succinate 
pH 7 and have been searching for suitable cryo conditions.
Is succinate in itself already a cryoprotectant 
(maybe at somewhat higher concentration) or should I

add e.g. glycerol ? Any experience or ideas are welcome.

Thanks in advance,


[]
   Ing. Tjaard Pijning

Protein Crystallography Group
University of Groningen
Nijenborgh 4
9747 AG Groningen
The Netherlands
Tel. (31)(0)50 363 4385





Dr. Alastair McEwen
Département de Biologie et Génomique Structurales
IGBMC, 1 rue Laurent Fries, BP10142
67404 ILLKIRCH, France
Tel:  +33 (0)3 88 65 57 73
Fax: +33 (0)3 88 65 32 76
email: [EMAIL PROTECTED] <>

Dr. Alastair McEwen
Département de Biologie et Génomique Structurales
IGBMC, 1 rue Laurent Fries, BP10142
67404 ILLKIRCH, France
Tel:  +33 (0)3 88 65 57 73
Fax: +33 (0)3 88 65 32 76
email: [EMAIL PROTECTED]

Re: [ccp4bb] Cryo Vs crystal size

[Jürgen Bosch]

There are a couple of additional factors not taken into account here.

1. LN2 versus frozen in strem or propane etc
2. did you try to flash anneal the larger crystal
3. smeary diffraction from the big crystal or not ?
4. how much residual solvent was around your crystal when freezing ?

In general smaller crystals are anyhow better in my hands.

Jürgen

On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:

> Hi All
> 
> I had two crystals grown in same well, one is small and other is 10 times 
> bigger. I treated both crystal in same cryo and same time. The smaller one 
> diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one 
> to diffract high resolution. 
> 
> I assume the bigger crystal might have lot of solvent which prevent for high 
> resolution. If it is true what could be the best way to dehydrate crystal 
> without affecting crystal quality?
> 
> Thank you
> 
> Syed
> 
> PS: Taken care of less solvent to be present in the loop
> 
> 
> 

-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] Cryo Vs crystal size

[W H]

50% solvent content in a small crystal is still 50% solvent content in
a large crystal given the same crystal form, so it's tough to use that
to explain loss of diffraction.

Very large crystals in my hands seem to suffer from more problems of
mechanical stress.



William


On Wed, Apr 14, 2010 at 5:36 PM, syed ibrahim  wrote:
> Hi All
>
> I had two crystals grown in same well, one is small and other is 10 times 
> bigger. I treated both crystal in same cryo and same time. The smaller one 
> diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one 
> to diffract high resolution.
>
> I assume the bigger crystal might have lot of solvent which prevent for high 
> resolution. If it is true what could be the best way to dehydrate crystal 
> without affecting crystal quality?
>
> Thank you
>
> Syed
>
> PS: Taken care of less solvent to be present in the loop
>
>
>
>

Re: [ccp4bb] Cryo Vs crystal size

[Roger Rowlett]

I agree. Large crystals often diffract more poorly than smaller ones.
I would hazard a guess that large crystals may experience more
mechanical distortion during flash cooling, but it's just a guess. In
our lab, the sweet spot for most of our crystals is 0.3-0.4 mm. Larger
is usually worse, and smaller has weaker scattering.

Cheers, Roger Rowlett

On 4/14/10, W H  wrote:
> 50% solvent content in a small crystal is still 50% solvent content in
> a large crystal given the same crystal form, so it's tough to use that
> to explain loss of diffraction.
>
> Very large crystals in my hands seem to suffer from more problems of
> mechanical stress.
>
>
>
> William
>
>
> On Wed, Apr 14, 2010 at 5:36 PM, syed ibrahim 
> wrote:
>> Hi All
>>
>> I had two crystals grown in same well, one is small and other is 10 times
>> bigger. I treated both crystal in same cryo and same time. The smaller one
>> diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger
>> one to diffract high resolution.
>>
>> I assume the bigger crystal might have lot of solvent which prevent for
>> high resolution. If it is true what could be the best way to dehydrate
>> crystal without affecting crystal quality?
>>
>> Thank you
>>
>> Syed
>>
>> PS: Taken care of less solvent to be present in the loop
>>
>>
>>
>>
>

-- 
Sent from my mobile device

Re: [ccp4bb] Cryo Vs crystal size

[Ho Leung Ng]

Like others have pointed out, I've often found very large crystals (> 0.4
mm) to not diffract as well, either due to growth defects or poor
cryoprotection. How large are the crystals you're talking about? You could
try chipping a piece off your large crystal and see how well that diffracts.


ho

Re: [ccp4bb] Cryo Vs crystal size

[syed ibrahim]

Dear Jurgen and Ho Leung

To add few more point regarding my question:

1. Crystal was first  frozen in LN2 and then transfered to cryo stream (in 
presence of LN2 in vial)
2. Anealing did not help (both short time and long time) -  perhaps the crystal 
dies.
3.  Spots are clear to available resolution (is:  6-7A). In the high resolution 
region there is no spot but looks like smear in the whole area.
4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on 0.5mm 
loop. So the liquid around the crystal was very less. I deliberately avoided 
more solvent in the loop to help diffraction.

Thanks

Syed



--- On Thu, 4/15/10, Jürgen Bosch  wrote:

From: Jürgen Bosch 
Subject: Re: [ccp4bb] Cryo Vs crystal size
To: CCP4BB@JISCMAIL.AC.UK
Date: Thursday, April 15, 2010, 3:46 AM

There are a couple of additional factors not taken into account here.
1. LN2 versus frozen in strem or propane etc2. did you try to flash anneal the 
larger crystal3. smeary diffraction from the big crystal or not ?4. how much 
residual solvent was around your crystal when freezing ?
In general smaller crystals are anyhow better in my hands.
Jürgen
On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:
Hi All

I had two crystals grown in same well, one is small and other is 10 times 
bigger. I treated both crystal in same cryo and same time. The smaller one 
diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one 
to diffract high resolution. 

I assume the bigger crystal might have lot of solvent which prevent for high 
resolution. If it is true what could be the best way to dehydrate crystal 
without affecting crystal quality?

Thank you

Syed

PS: Taken care of less solvent to be present in the loop





-Jürgen BoschJohns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/





  

Re: [ccp4bb] Cryo Vs crystal size

[Anastassis Perrakis]

Hi -

My two cents:

First, you say:

I assume the bigger crystal might have lot of solvent which prevent  
for high resolution. If it is true what could be the best way to  
dehydrate crystal without affecting crystal quality?


I think this assumption is confusing. If the crystals were grown in  
the same drop/condition, they have identical percentage solvent  
content. Thus, you do not want to look at dehydration, the 'percentage  
solvent content' is fine. What you want to look at is the mechanics of  
vitrification. Big crystals, are simply hard to freeze: because of  
their volume they cannot be vitrified as rapidly and uniformly as  
smaller crystals. I will not be surprised if there are papers that  
quantify that, but what I am saying here is only from experience and  
adding a 'logical' explanation to that experience.


Thus, I would simply stay with the smaller crystals (I have a feeling  
that you 'small' crystals are 'big' for many other people) and be  
happy they diffract to 2.5 A (is that SR or RA?)


A.


On Apr 15, 2010, at 3:16, syed ibrahim wrote:



Dear Jurgen and Ho Leung

To add few more point regarding my question:

1. Crystal was first  frozen in LN2 and then transfered to cryo  
stream (in presence of LN2 in vial)
2. Anealing did not help (both short time and long time) -  perhaps  
the crystal dies.
3.  Spots are clear to available resolution (is:  6-7A). In the high  
resolution region there is no spot but looks like smear in the whole  
area.
4. The crystal was approximately 1.0mm length and 0.4mm dia. I  
mounted on 0.5mm loop. So the liquid around the crystal was very  
less. I deliberately avoided more solvent in the loop to help  
diffraction.


Thanks

Syed



--- On Thu, 4/15/10, Jürgen Bosch  wrote:

From: Jürgen Bosch 
Subject: Re: [ccp4bb] Cryo Vs crystal size
To: CCP4BB@JISCMAIL.AC.UK
Date: Thursday, April 15, 2010, 3:46 AM

There are a couple of additional factors not taken into account here.

1. LN2 versus frozen in strem or propane etc
2. did you try to flash anneal the larger crystal
3. smeary diffraction from the big crystal or not ?
4. how much residual solvent was around your crystal when freezing ?

In general smaller crystals are anyhow better in my hands.

Jürgen

On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:


Hi All

I had two crystals grown in same well, one is small and other is 10  
times bigger. I treated both crystal in same cryo and same time.  
The smaller one diffracted to 2.5A and the bigger one to 6-7A. I  
was expecting the bigger one to diffract high resolution.


I assume the bigger crystal might have lot of solvent which prevent  
for high resolution. If it is true what could be the best way to  
dehydrate crystal without affecting crystal quality?


Thank you

Syed

PS: Taken care of less solvent to be present in the loop





-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/




P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Cryo Vs crystal size

[Mark J. van Raaij]

and don't forget to check diffraction without freezing.
Mark

On 15 April 2010 10:37, Anastassis Perrakis  wrote:

> Hi -
>
> My two cents:
>
> First, you say:
>
> I assume the bigger crystal might have lot of solvent which prevent for
> high resolution. If it is true what could be the best way to dehydrate
> crystal without affecting crystal quality?
>
>
> I think this assumption is confusing. If the crystals were grown in the
> same drop/condition, they have identical percentage solvent content. Thus,
> you do not want to look at dehydration, the 'percentage solvent content' is
> fine. What you want to look at is the mechanics of vitrification. Big
> crystals, are simply hard to freeze: because of their volume they cannot be
> vitrified as rapidly and uniformly as smaller crystals. I will not be
> surprised if there are papers that quantify that, but what I am saying here
> is only from experience and adding a 'logical' explanation to that
> experience.
>
> Thus, I would simply stay with the smaller crystals (I have a feeling that
> you 'small' crystals are 'big' for many other people) and be happy they
> diffract to 2.5 A (is that SR or RA?)
>
> A.
>
>
> On Apr 15, 2010, at 3:16, syed ibrahim wrote:
>
>
> Dear Jurgen and Ho Leung
>
> To add few more point regarding my question:
>
> 1. Crystal was first  frozen in LN2 and then transfered to cryo stream (in
> presence of LN2 in vial)
> 2. Anealing did not help (both short time and long time) -  perhaps the
> crystal dies.
> 3.  Spots are clear to available resolution (is:  6-7A). In the high
> resolution region there is no spot but looks like smear in the whole area.
> 4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on
> 0.5mm loop. So the liquid around the crystal was very less. I deliberately
> avoided more solvent in the loop to help diffraction.
>
> Thanks
>
> Syed
>
>
>
> --- On *Thu, 4/15/10, Jürgen Bosch * wrote:
>
>
> From: Jürgen Bosch 
> Subject: Re: [ccp4bb] Cryo Vs crystal size
> To: CCP4BB@JISCMAIL.AC.UK
> Date: Thursday, April 15, 2010, 3:46 AM
>
> There are a couple of additional factors not taken into account here.
>
> 1. LN2 versus frozen in strem or propane etc
> 2. did you try to flash anneal the larger crystal
> 3. smeary diffraction from the big crystal or not ?
> 4. how much residual solvent was around your crystal when freezing ?
>
> In general smaller crystals are anyhow better in my hands.
>
> Jürgen
>
> On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:
>
> Hi All
>
> I had two crystals grown in same well, one is small and other is 10 times
> bigger. I treated both crystal in same cryo and same time. The smaller one
> diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger
> one to diffract high resolution.
>
> I assume the bigger crystal might have lot of solvent which prevent for
> high resolution. If it is true what could be the best way to dehydrate
> crystal without affecting crystal quality?
>
> Thank you
>
> Syed
>
> PS: Taken care of less solvent to be present in the loop
>
>
>
>
> -
> Jürgen Bosch
> Johns Hopkins Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Phone: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-3655
> http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/>
>
>
>
> *P** **please don't print this e-mail unless you really need to*
> Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
> Department of Biochemistry (B8)
> Netherlands Cancer Institute,
> Dept. B8, 1066 CX Amsterdam, The Netherlands
> Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
>
>
>
>
>


-- 
Mark J van Raaij
http://webspersoais.usc.es/mark.vanraaij
http://www.ibmb.csic.es

Re: [ccp4bb] Cryo Vs crystal size

[Andy Torelli]

You've gotten some helpful replies already.  I have found the following 
reference to be helpful in understanding some of the physics behind 
damage incurred during the crystal cooling process and a general 
strategy to help avoid it.  It expands upon what's already been said - 
that larger crystals are more prone to distress during cooling.  This 
and other papers from the same group contain useful information and advice.


A General Method for Hyperquenching Protein Crystals
Matthew Warkentin and Robert E. Thorne
Struct Funct Genomics. 2007 December ; 8(4): 141–144. 
doi:10.1007/s10969-007-9029-0.


Best,
-Andy


On 4/15/2010 4:48 AM, Mark J. van Raaij wrote:

and don't forget to check diffraction without freezing.
Mark

On 15 April 2010 10:37, Anastassis Perrakis mailto:a.perra...@nki.nl>> wrote:

Hi -

My two cents:

First, you say:


I assume the bigger crystal might have lot of solvent which
prevent for high resolution. If it is true what could be the
best way to dehydrate crystal without affecting crystal quality?




I think this assumption is confusing. If the crystals were grown in
the same drop/condition, they have identical percentage solvent
content. Thus, you do not want to look at dehydration, the
'percentage solvent content' is fine. What you want to look at is
the mechanics of vitrification. Big crystals, are simply hard to
freeze: because of their volume they cannot be vitrified as rapidly
and uniformly as smaller crystals. I will not be surprised if there
are papers that quantify that, but what I am saying here is only
from experience and adding a 'logical' explanation to that experience.

Thus, I would simply stay with the smaller crystals (I have a
feeling that you 'small' crystals are 'big' for many other people)
and be happy they diffract to 2.5 A (is that SR or RA?)

A.


On Apr 15, 2010, at 3:16, syed ibrahim wrote:



Dear Jurgen and Ho Leung

To add few more point regarding my question:

1. Crystal was first  frozen in LN2 and then transfered to cryo
stream (in presence of LN2 in vial)
2. Anealing did not help (both short time and long time) -
perhaps the crystal dies.
3.  Spots are clear to available resolution (is:  6-7A). In the
high resolution region there is no spot but looks like smear in
the whole area.
4. The crystal was approximately 1.0mm length and 0.4mm dia. I
mounted on 0.5mm loop. So the liquid around the crystal was very
less. I deliberately avoided more solvent in the loop to help
diffraction.

Thanks

Syed



--- On *Thu, 4/15/10, Jürgen Bosch /mailto:jubo...@jhsph.edu>>/* wrote:


    From: Jürgen Bosch mailto:jubo...@jhsph.edu>>
Subject: Re: [ccp4bb] Cryo Vs crystal size
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Date: Thursday, April 15, 2010, 3:46 AM

There are a couple of additional factors not taken into
account here.

1. LN2 versus frozen in strem or propane etc
2. did you try to flash anneal the larger crystal
3. smeary diffraction from the big crystal or not ?
4. how much residual solvent was around your crystal when
freezing ?

In general smaller crystals are anyhow better in my hands.

Jürgen

On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:


Hi All

I had two crystals grown in same well, one is small and other
is 10 times bigger. I treated both crystal in same cryo and
same time. The smaller one diffracted to 2.5A and the bigger
one to 6-7A. I was expecting the bigger one to diffract high
resolution.

I assume the bigger crystal might have lot of solvent which
prevent for high resolution. If it is true what could be the
best way to dehydrate crystal without affecting crystal quality?

Thank you

Syed

PS: Taken care of less solvent to be present in the loop





-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/>




*P** **please don't print this e-mail unless you really need to*
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791







--
Mark J van Raaij
http://webspersoais.usc.es/mark.vanraaij
http://www.ibmb.csic.es

Re: [ccp4bb] cryo-em data conversion

[Kabasakal, Burak V]

You can convert it using EMAN2 by e2proc2d.py by typing

e2proc2d.py *.tif @.mrc


.star file can be generated in Relion.


Also, you may subscribe the mailing list CCP-EM for EM-related questions.

  https://www.jiscmail.ac.uk/CCPEM.

JISCMail - CCPEM List at WWW.JISCMAIL.AC.UK
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Regards,


Burak


From: CCP4 bulletin board  on behalf of Hena Dutta 

Sent: 08 February 2018 20:46:54
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo-em data conversion

Dear Members,
Sorry for if the question is off topic. Can someone suggest the easiest way to 
convert .tif file (raw data) into .mrcs format and also need to create a .star 
file to use Relion for the following steps?
Thanks for your help.

Best regards,
Hena

Re: [ccp4bb] cryo was:bigger size - > better diffraction?

[Tommi Kajander]

for cryoprotection just drag it through paratone-N (put next to your crystal
drop) and you dont have to  worry about ill effects on the crystal
(usually). this will cut down the long road with cryoprotectant search to
minimal. (..not sure if this had anything to do with the original topic, but
since it keeps coming up...) it doesnt penetrate the crystal and works and
you dont have to  waste time and crystals on finding a solution with some
soluble cryoprotection additive that doenst kill the crystal.. 

tommi

Quoting Leonard Thomas <[EMAIL PROTECTED]>:

> The first thing to try before going down the long road of fussing  
> with cryo is to take a shot at room temp. and see how your crystal  
> diffracts in general.  It is true it may be a cryo problem, but if  
> the non cryo protected crystals do not diffract then why would one  
> expect the cryo protected one to.
> 
> As for controlling crystal growth.  I would second what Shane wrote  
> and try seeding.   Also trying the usually additives and varying  
> protein concentration/precipitant concentration should help also so.
> 
> Len
> 
> 
> On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote:
> 
> > Streak seeding all your trays should give you a better handle on
> > nucleation. You might be too high in protein or precipitant w/o the
> > seeding, hence the showering and rare nice crystals.
> >
> > Varying cryos, or cryo concentrations, or how the cryo is added can  
> > help
> > a lot. Try 5 or 6 different cryos at 3 concentrations each. When you
> > have the best nailed down then try sequential transfers of the  
> > crystals
> > from low to target concentrations (e.g. into 5% for a couple minutes,
> > then 10, 20, 25%). Also, you can try growing the crystals w/ a bit  
> > (5%)
> > or the final conc or cryo already present. Should help in getting them
> > habituated to the cryo.
> >
> > Shane Atwell
> >
> >> -Original Message-
> >> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
> >> Behalf Of Jenny
> >> Sent: Wednesday, April 04, 2007 5:50 AM
> >> To: CCP4BB@JISCMAIL.AC.UK
> >> Subject: [ccp4bb] bigger size - > better diffraction?
> >>
> >> Hi, All,
> >>
> >> I got a crystal that diffracts at 3.3A in house.The crystal
> >> size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the
> >> size is fine,but it turns out the smaller ones diffract
> >> worse.I guess the reason is that
> >> the cell unit is really big (126.292   126.292   134.904  p4212,
> >> pretty big for a 10kD protein, isn't it?)
> >>
> >> So looks like I need to grow bigger crystals in order to get
> >> better diffractions.The problems is ,every time when I set up
> >> trays, the growing conditions is not exactly the same, so I
> >> have to set up a whole tray or maybe even 2 trays , then 2 or
> >> 3 conditions will jump out with good crystals ( 2 or 3
> >> nucleation site ) and some of the others will show lots lots
> >> of small crystals.I used NaCl as the salt, in a 4*6 tray, the
> >> [NaCl] is going from 2.0,2.05,2.1,2.15,something like
> >> that and 0.05M does make big difference.I used Urea as the
> >> additive in this case ( 25 m ~  100 mM) and tried
> >> 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better
> >> than the other two cases.Right now it's growing in room temp
> >> in about a week.And crystals that not fresh got some bubbles
> >> around the edge and didn't diffract well.
> >>
> >> Does anyone have any suggestions that what I could do to
> >> improve the diffraction?
> >>
> >> Thanks a lot.
> >>
> >> Jenny
> >>
> 


-- 
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
PO box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax  +358-9-191 59940

Re: [ccp4bb] cryo for high salt crystal

[conan仙人指路]

Min,
In some of my Xtal conditions, I only have 1.5 to 2M AmSO4 without KCl, the 
salt comes out of solution only after maybe several minutes which is enough to 
harvest the crystal. Maybe you could just direct freeze to try the diffraction 
since it is already at high salt concentration or maybe add some mother liquor 
to prevent it the salting out. I also suggest Mitegen LV cryo oil which works 
for many cases for me.
Best,Hongnan Cao, Ph.D.Department of BiochemistryGreat Lakes Bioenergy Research 
CenterUniversity of Wisconsin, Madison
Date: Tue, 10 Jul 2012 12:28:30 -0400
From: mzhang...@hotmail.com
Subject: [ccp4bb] cryo for high salt crystal
To: CCP4BB@JISCMAIL.AC.UK





regaentDear All,
I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M 
Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, 
paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo 
plus original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am trying to 
loop the crystal, both the drop and cryoprotectant drop form salt crystals (not 
sure it is KCl or ammonia sulfate) significantly and very quickly, that cause 
my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does 
anyone here have similiar case? any suggestion will be appreciated.
Thanks,Min  
  

Re: [ccp4bb] cryo for high salt crystal

[Christian Roth]

Hi,

I have used pure malonate (above 3M) for a similar condition. Sometimes the 
cryoprotectant alone, without reservoir works quite well

Cheers 

Christian 
 
Am Dienstag 10 Juli 2012 18:28:30 schrieb m zhang:
> regaentDear All,
> I am sure this question was discussed before. But I am wondering if anyone
>  got the same experience as I do. I got a crystal out of condition with 1M
>  KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene
>  glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The
>  problem is that all the cryo plus original reagents in the reservoir
>  precipitate the salts out. And more serious problem is because of high
>  salt in the condition, while I am trying to loop the crystal, both the
>  drop and cryoprotectant drop form salt crystals (not sure it is KCl or
>  ammonia sulfate) significantly and very quickly, that cause my crystal
>  dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone
>  here have similiar case? any suggestion will be appreciated. Thanks,Min
> 

Re: [ccp4bb] cryo for high salt crystal

[David Schuller]

Pressure cryocooling lowers the need for cryoprotectant dramatically.


On 07/10/12 12:28, m zhang wrote:

regaentDear All,

I am sure this question was discussed before. But I am wondering if 
anyone got the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at 
pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N 
oil, or ammonium sulfate itself: The problem is that all the cryo plus 
original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am 
trying to loop the crystal, both the drop and cryoprotectant drop form 
salt crystals (not sure it is KCl or ammonia sulfate) significantly 
and very quickly, that cause my crystal dissolved. My crystal doesn't 
seem to survive paraton-N oil. Does anyone here have similiar case? 
any suggestion will be appreciated.


Thanks,
Min



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

Re: [ccp4bb] cryo for high salt crystal

[Jim Pflugrath]

Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or 
v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 
50% saturated in reservoir.  You will have to TEST these.  See also this 
webinar on cryocrystallography which shows how to make these solutions: 
http://www.rigaku.com/node/1388

You could also try high salt solutions with similar technique.

Good luck!

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang 
[mzhang...@hotmail.com]
Sent: Tuesday, July 10, 2012 11:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo for high salt crystal

regaentDear All,

I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I 
tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium 
sulfate itself: The problem is that all the cryo plus original reagents in the 
reservoir precipitate the salts out. And more serious problem is because of 
high salt in the condition, while I am trying to loop the crystal, both the 
drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia 
sulfate) significantly and very quickly, that cause my crystal dissolved. My 
crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar 
case? any suggestion will be appreciated.

Thanks,
Min

Re: [ccp4bb] cryo for high salt crystal

[James Holton]

A nice review of "cryosalts" is available here:
http://dx.doi.org/10.1107/S090744497587

For crystals that "don't seem to survive paratone-N", I recommend using 
a microscope equipped with a polarizer.  The refractive index of 
paratone-N is much closer to that of protein crystals than that of 
water, so one can easily be fooled into thinking the crystal "dissolved" 
when what you are actually seeing is the "disappearing pyrex effect":

https://sites.google.com/site/rooseveltphysics/pyrexandveggieoil.jpg
A nice example of the "disappearing crystal" due to refractive index 
matching is also apparent in figure 1d of:

http://dx.doi.org/10.1107/S0021889809023553
Sadly, I think many crystallographers have abandoned my favorite oil 
because they didn't know there crystal was actually still intact and happy!


As for salt crystals growing while you are trying to harvest: yes, this 
is annoying.  This is why I usually bury my drops in paratone-N oil 
immediately after opening them.  It slows down evaporation 
tremendously.  If you don't like working through oil, then you must 
either work very quickly or find a way to humidify the atmosphere around 
your drop after you open it.  You can spend an arbitrary amount of money 
on "wet air machines" these days, but if you happen to know what your 
reservoir contains (you should), and you don't mind making up a liter or 
two of it, then all you need to do is bubble air or N2 through a big vat 
of your "reservoir solution" and then direct that bubbled-off gas 
through a pipe at your drop.  I imagine this would be a great way to 
deal with alcohol-based conditions as well (provided you had adequate 
ventilation).


-James Holton
MAD Scientist


On 7/10/2012 9:28 AM, m zhang wrote:

regaentDear All,

I am sure this question was discussed before. But I am wondering if 
anyone got the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at 
pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N 
oil, or ammonium sulfate itself: The problem is that all the cryo plus 
original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am 
trying to loop the crystal, both the drop and cryoprotectant drop form 
salt crystals (not sure it is KCl or ammonia sulfate) significantly 
and very quickly, that cause my crystal dissolved. My crystal doesn't 
seem to survive paraton-N oil. Does anyone here have similiar case? 
any suggestion will be appreciated.


Thanks,
Min

Re: [ccp4bb] cryo for high salt crystal

[anna anna]

Hi Min,
I agree with Christian Roth, try malonate at high concentration, 2.8 molar
is enough. I suggest to adjust the pH to about the value of your
xtallization condition.
To avoyd salt xtals formation, humidify the environment, some wet paper
around the drop can do the job!

cheers
anna


2012/7/10 m zhang 

>  regaentDear All,
>
> I am sure this question was discussed before. But I am wondering if anyone
> got the same experience as I do.
> I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at
> pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil,
> or ammonium sulfate itself: The problem is that all the cryo plus original
> reagents in the reservoir precipitate the salts out. And more serious
> problem is because of high salt in the condition, while I am trying to loop
> the crystal, both the drop and cryoprotectant drop form salt crystals (not
> sure it is KCl or ammonia sulfate) significantly and very quickly, that
> cause my crystal dissolved. My crystal doesn't seem to survive paraton-N
> oil. Does anyone here have similiar case? any suggestion will be
> appreciated.
>
> Thanks,
> Min
>

Re: [ccp4bb] cryo for high salt crystal

[Craig Bingman]

You can sometimes cryoprotect crystals like this with the Mitegen low viscosity 
cryo oil and their micro mount loops.  If that doesn't work, then you can 
gradually raise the concentration of sucrose or ethylene glycol so that your 
crystals freeze well.  I don't think it should take anything like 25% sucrose 
to cryoprotect this in a small mount with little loose solvent around the 
crystal.  Probably less than 10% would do the trick if you can really wick away 
most of the liquid and get it into the liquid nitrogen as rapidly as possible.  
Robert Thorne's lab at Cornell has been important in exploring this 
cryoprotection space.  

(from the lab web page)

Effects of cryoprotectant concentration and cooling rate on vitrification of 
aqueous solutions. V. Berejnov, N. S. Husseini, O. A., Alsaied and R . E. 
Thorne, J. Appl. Cryst. 39, 244-251 (2006).
Hyperquenching for Protein Crystallography. M. Warkentin, V. Berejnov, and R. 
E. Thorne, J. Appl. Cryst. J. Appl. Cryst. 39, 805-811 (2006).
Cryocrystallography in capillaries: critical glycerol concentrations and 
cooling rates. M. Warkentin, V. Stanislavskaia, K. Hammes and R. E. Thorne, J. 
Appl. Cryst. 41, 791-797 (2008).


On Jul 10, 2012, at 11:50 AM, Christian Roth wrote:

> Hi,
> 
> I have used pure malonate (above 3M) for a similar condition. Sometimes the 
> cryoprotectant alone, without reservoir works quite well
> 
> Cheers 
> 
> Christian 
> 
> Am Dienstag 10 Juli 2012 18:28:30 schrieb m zhang:
>> regaentDear All,
>> I am sure this question was discussed before. But I am wondering if anyone
>> got the same experience as I do. I got a crystal out of condition with 1M
>> KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene
>> glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The
>> problem is that all the cryo plus original reagents in the reservoir
>> precipitate the salts out. And more serious problem is because of high
>> salt in the condition, while I am trying to loop the crystal, both the
>> drop and cryoprotectant drop form salt crystals (not sure it is KCl or
>> ammonia sulfate) significantly and very quickly, that cause my crystal
>> dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone
>> here have similiar case? any suggestion will be appreciated. Thanks,Min
>> 

Re: [ccp4bb] cryo for high salt crystal

[m zhang]

Hi Jim,
25% is w/v. Thanks for the information. Will check the webinar.
Thanks,Min

From: jim.pflugr...@rigaku.com
To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] cryo for high salt crystal
Date: Tue, 10 Jul 2012 17:39:56 +







Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or 
v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 
50% saturated in reservoir.
  You will have to TEST these.  See also this webinar on cryocrystallography 
which shows how to make these solutions: http://www.rigaku.com/node/1388



You could also try high salt solutions with similar technique.




Good luck!



Jim










From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang 
[mzhang...@hotmail.com]

Sent: Tuesday, July 10, 2012 11:28 AM

To: CCP4BB@JISCMAIL.AC.UK

Subject: [ccp4bb] cryo for high salt crystal






regaentDear All,



I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do. 
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I 
tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium 
sulfate itself: The problem is that all the cryo plus original reagents in the 
reservoir precipitate
 the salts out. And more serious problem is because of high salt in the 
condition, while I am trying to loop the crystal, both the drop and 
cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) 
significantly and very quickly, that cause
 my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does 
anyone here have similiar case? any suggestion will be appreciated.



Thanks,
Min








  

Re: [ccp4bb] cryo for high salt crystal

[Roger Rowlett]

We frequently crystallize one of our proteins and variants of it in 
1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% 
glycerol or 25-30% glucose does not cause precipitation of salts. Both 
KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in 
water, so I would not expect cryoprotectant concentrations of glycerol 
or glucose to cause precipitation (We can save cryoprotectant solutions 
of at least 2 M ammonium sulfate indefinitely). How are you introducing 
cryprotectant? We use one of two methods:


1. Fish the crystal out of the mother liquor and place into artificial
   mother liquor with the same composition as the well solution +
   cryoprotectant. For glycerol or other liquids, you have to make this
   from scratch. For glucose, we just weigh out 300 mg of glucose in a
   microcentrifuge tube and make to the 1.0 mL mark with well solution.
   (Mix well of course before use. Gentle heating in a block or
   sonication will help dissolve the glucose.
2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to
   the drop the crystals are in. You can do this all at once, or in
   stages, keeping the drop hydrated by placing the hanging drop back
   in the well between additions.

If your drops are drying out during crystal harvesting (very possible in 
dry conditions), you might try harvesting in the cold room, where 
evaporation is slower. We often have problems with crystal cracking and 
drop-drying in the winter months when the humidity is very low indoors. 
The cold room is usually humid enough and cold enough to slow 
evaporation to allow crystal harvesting. (I hate working in the meat 
locker, though.)


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu



On 7/12/2012 12:55 PM, m zhang wrote:

Hi Jim,

25% is w/v. Thanks for the information. Will check the webinar.

Thanks,
Min


From: jim.pflugr...@rigaku.com
To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] cryo for high salt crystal
Date: Tue, 10 Jul 2012 17:39:56 +

Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is 
that w/v or v/w?), try using 100% saturated in reservoir, 75% 
saturated in reservoir, or 50% saturated in reservoir.  You will have 
to TEST these.  See also this webinar on cryocrystallography which 
shows how to make these solutions: http://www.rigaku.com/node/1388


You could also try high salt solutions with similar technique.

Good luck!

Jim



*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m 
zhang [mzhang...@hotmail.com]

*Sent:* Tuesday, July 10, 2012 11:28 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] cryo for high salt crystal

regaentDear All,

I am sure this question was discussed before. But I am wondering if 
anyone got the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at 
pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N 
oil, or ammonium sulfate itself: The problem is that all the cryo plus 
original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am 
trying to loop the crystal, both the drop and cryoprotectant drop form 
salt crystals (not sure it is KCl or ammonia sulfate) significantly 
and very quickly, that cause my crystal dissolved. My crystal doesn't 
seem to survive paraton-N oil. Does anyone here have similiar case? 
any suggestion will be appreciated.


Thanks,
Min

Re: [ccp4bb] cryo for high salt crystal

[R. M. Garavito]

Roger's note reminded me of some older literature (old in the sense that this 
problem extends back into the mid-1970's).  Dealing with cryopreservation of 
crystals grown in "high" salt can be a real problem, but as many people have 
pointed out, the normal cryoprotectants can work, although many salts work just 
as well (not only malonate, but ammonium formate, lithium citrate, etc.).  I 
don't consider 1.6-2 M ammonium sulfate as really high salt; try working with 3 
M ammonium sulfate.  However, the trick is not simply the cryoprotectant, but 
also the exchange method, as Roger mentions.  While you can make artificial 
mother liquors (I love these old terms) with high concentrations of salt and 
nonionic cryoprotectants (sugars, glycerol, ethylene glycol, etc.), that does 
not mean they will readily exchange with the crystal, even after soaking for 
hours.  

Bill Ray, a classical enzymologist and physical biochemist from Purdue, really 
wanted to determine the crystal structure of phosphoglucomutase with ligands, 
but there were many difficulties.  Using crystals grown in 2.1 M ammonium 
sulfate was one of these problems.  He realized that the phase interactions 
between the bulk solution (i.e., the mother liquor) and the interstitial 
salt-rich solvent was a major obstacle in the proper solvent exchange and good 
subsequent cryopreservation. See how he solved the problem:

W. J. Ray, Jr., et al. Removal of salt from a salt-induced protein crystal 
without cross-linking. Preliminary examination of "desalted" crystals of 
phosphoglucomutase by X-ray crystallography at low temperature. Biochemistry. 
1991 Jul 16;30(28):6866-75.

Cheers,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On Jul 12, 2012, at 2:10 PM, Roger Rowlett wrote:

> We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M 
> ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% 
> glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium 
> sulfate (5.6 M) have enormous solubilities in water, so I would not expect 
> cryoprotectant concentrations of glycerol or glucose to cause precipitation 
> (We can save cryoprotectant solutions of at least 2 M ammonium sulfate 
> indefinitely). How are you introducing cryprotectant? We use one of two 
> methods:
> 
> Fish the crystal out of the mother liquor and place into artificial mother 
> liquor with the same composition as the well solution + cryoprotectant. For 
> glycerol or other liquids, you have to make this from scratch. For glucose, 
> we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 
> 1.0 mL mark with well solution. (Mix well of course before use. Gentle 
> heating in a block or sonication will help dissolve the glucose.
> Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop 
> the crystals are in. You can do this all at once, or in stages, keeping the 
> drop hydrated by placing the hanging drop back in the well between additions.
> If your drops are drying out during crystal harvesting (very possible in dry 
> conditions), you might try harvesting in the cold room, where evaporation is 
> slower. We often have problems with crystal cracking and drop-drying in the 
> winter months when the humidity is very low indoors. The cold room is usually 
> humid enough and cold enough to slow evaporation to allow crystal harvesting. 
> (I hate working in the meat locker, though.)
> Cheers,
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
> 
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
> 
> 
> On 7/12/2012 12:55 PM, m zhang wrote:
>> Hi Jim,
>> 
>> 25% is w/v. Thanks for the information. Will check the webinar.
>> 
>> Thanks,
>> Min
>> 
>> From: jim.pflugr...@rigaku.com
>> To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
>> Subject: RE: [ccp4bb] cryo for high salt crystal
>> Date: Tue, 10 Jul 2012 17:39:56 +
>> 
>> Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v 
>> or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, 
>> or 50% saturated in reservoir.  You will have to TEST these.  See also this 
>> webinar on cryocrystallograph

Re: [ccp4bb] cryo for high salt crystal

[Anna Gardberg]

Hello, Min.
Lithium sulfate is a cryoprotectant at 2.0 M and sometimes even less (much
lower than the concentrations needed for ammonium sulfate), so I would try
replacing your ammonium sulfate with lithium sulfate, creating a
cryoprotectant with 0.5-1.0 M KCl, 1.4-2.0 M LiSO4, at pH 7. You might need
to transfer your crystals through a couple of intermediate drops.

Regarding the reservoir precipitation - is there any chance you could
control the humidity of the area in which you're working? Even filling a
couple of adjacent reservoirs with water might help buy you a few extra
crucial seconds. Also, working in a cold room to harvest your crystals will
help reduce the evaporation rate.

Good luck!

Best,
Anna

On Tue, Jul 10, 2012 at 9:28 AM, m zhang  wrote:

>  regaentDear All,
>
> I am sure this question was discussed before. But I am wondering if anyone
> got the same experience as I do.
> I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at
> pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil,
> or ammonium sulfate itself: The problem is that all the cryo plus original
> reagents in the reservoir precipitate the salts out. And more serious
> problem is because of high salt in the condition, while I am trying to loop
> the crystal, both the drop and cryoprotectant drop form salt crystals (not
> sure it is KCl or ammonia sulfate) significantly and very quickly, that
> cause my crystal dissolved. My crystal doesn't seem to survive paraton-N
> oil. Does anyone here have similiar case? any suggestion will be
> appreciated.
>
> Thanks,
> Min
>

Re: [ccp4bb] cryo for high salt crystal

[m zhang]

Hi Anna,
Actually I just tried lithium sulfate: I made a cryo with 1M KCl, 1.3M Lithium 
sulfate to replace the ammonia sulfate totally at pH7. it is not freezing 
clear. I guess I should try to increase the amount of LiSO4. But from all the 
warm responses here, I found sometimes people don't necessary keep the same 
concentration of component in the cryo as in the well solution + 
cryoprotectant. I usually make the artificial mother liquor as Roger or Michael 
mentioned early on. So my question is: will that hurt the crystal if the 
concentration of the component of cryo change?
Want to thanks to all for your warm suggestions. I haven't finish all the 
reading suggested here. but will.
Thanks,Manqing

Date: Thu, 12 Jul 2012 13:40:39 -0700
From: anna.s.gardb...@gmail.com
Subject: Re: [ccp4bb] cryo for high salt crystal
To: CCP4BB@JISCMAIL.AC.UK

Hello, Min.Lithium sulfate is a cryoprotectant at 2.0 M and sometimes even less 
(much lower than the concentrations needed for ammonium sulfate), so I would 
try replacing your ammonium sulfate with lithium sulfate, creating a 
cryoprotectant with 0.5-1.0 M KCl, 1.4-2.0 M LiSO4, at pH 7. You might need to 
transfer your crystals through a couple of intermediate drops. 

Regarding the reservoir precipitation - is there any chance you could control 
the humidity of the area in which you're working? Even filling a couple of 
adjacent reservoirs with water might help buy you a few extra crucial seconds. 
Also, working in a cold room to harvest your crystals will help reduce the 
evaporation rate.

Good luck!
Best,Anna

On Tue, Jul 10, 2012 at 9:28 AM, m zhang  wrote:





regaentDear All,
I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M 
Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, 
paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo 
plus original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am trying to 
loop the crystal, both the drop and cryoprotectant drop form salt crystals (not 
sure it is KCl or ammonia sulfate) significantly and very quickly, that cause 
my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does 
anyone here have similiar case? any suggestion will be appreciated.

Thanks,Min


  

Re: [ccp4bb] Cryo-em model building with Balbes

[cheng tat cheung]

phenix.map_to_structure_factors can do the conversion, and then all the 
crystallographic tools for model building should be ready to use.

Tat

Sent from my iPhone

> On 25 Apr 2017, at 10:36 AM, KL Ho 
> <05c3a58110fa-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Dear All,
> 
> We are interested to build an atomic model on a 3.7 A cryo-em map with 
> BALBES-MOLREP pipeline. The map is currently in mrc format. It cannot be read 
> by BALBES (the error message is structure factor file size exceed limit). I 
> was wondering should I convert the mrc file to mtz format? Any idea how to 
> convert the mrc to mtz? 
> 
> Many thanks in advance. 
> 
> Best wishes
> Kok-Lian

Re: [ccp4bb] Cryo-em model building with Balbes

[Martyn Winn]

Hi,

MRC is a map format, and doesn't hold the reciprocal space structure factors 
that Balbes expects, and that are referred to in the error message. Yes, you 
will need to calculate structure factors, which are held in the MTZ format. 

Simplest way is to use Refmac, which deals with issues like box size and map 
sharpening. See step 1) described at   
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#id.seab0879x62k
 

More options for model fitting, building and refinement are on the CCP-EM page 
for the recent Icknield workshop:  
http://www.ccpem.ac.uk/training/icknield_2017/icknield_2017.php

HTH
m

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of cheng tat 
cheung
Sent: 25 April 2017 16:18
To: ccp4bb
Subject: Re: [ccp4bb] Cryo-em model building with Balbes

phenix.map_to_structure_factors can do the conversion, and then all the 
crystallographic tools for model building should be ready to use.

Tat

Sent from my iPhone

> On 25 Apr 2017, at 10:36 AM, KL Ho 
> <05c3a58110fa-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Dear All,
> 
> We are interested to build an atomic model on a 3.7 A cryo-em map with 
> BALBES-MOLREP pipeline. The map is currently in mrc format. It cannot be read 
> by BALBES (the error message is structure factor file size exceed limit). I 
> was wondering should I convert the mrc file to mtz format? Any idea how to 
> convert the mrc to mtz? 
> 
> Many thanks in advance. 
> 
> Best wishes
> Kok-Lian

Re: [ccp4bb] cryo-protection for crystals grown in ethanol

[Katya Heldwein]

Hi Rongjin,

I had crystals that grew in 9% ethanol as a precipitant. To cryoprotect
them, I first replaced the mother liquor with solution containing 10% MPD,
instead of ethanol, and then briefly dunked the crystals into ~30% MPD.

Hope this helps. Good luck.

Katya

On Mon, Sep 29, 2008 at 1:24 PM, Rongjin Guan <[EMAIL PROTECTED]> wrote:

>  Dear All
>
> I got crystals from 20% Ethanol with 0.1M Tris pH 8.5. This is my first
> time to have crystals in Ethanol and want to get some suggestions of
> cryo-protection from those who have done this before.
>
> I am waiting for my time on home X-ray facility, and hope I can get
> some suggestions before that.
>
> Thanks,
>
> Rongjin Guan
>

Re: [ccp4bb] cryo-protection for crystals grown in ethanol

[Mark J. van Raaij]

Dear Rongjin,

I would:
-prepare different cryosolutions, adding glycerol, replacing water  
with glycerol, replacing ethanol with other more cryogenic alcohols:  
if you have enough crystals, see how these behave when transferred to  
these solutions, if they crack, decrease precipitant, if they  
dissolve, increase.

once you have measurement time:
-first measure a couple of crystals in a capillary or mitegen "loop  
and sleeve" at room temperature to get a "best case" diffraction. On a  
home source you might even get a dataset, but surely you'll get good  
estimates of mosaic spread, spacegroup, and other crystallographic  
parameters, like for instance an idea about if the crystals may be  
twinned.
-then measure a couple of crystals directly frozen from the drop (i.e.  
"worst-case" scenario).
-then go for complete cryoprotected datasets, using the combined  
knowledge of previous experiments.
Even if the upcoming "beamtime" is not enough for all of this and you  
have to book more time in the coming weeks, I would go through all  
these steps and resist the temptation to go for "quick and dirty". If  
you only ever measure at 100K you never know how good the crystals  
were before freezing.


Mark

Quoting Rongjin Guan <[EMAIL PROTECTED]>


Dear All

I got crystals from 20% Ethanol with 0.1M Tris pH 8.5. This is my first
time to have crystals in Ethanol and want to get some suggestions of
cryo-protection from those who have done this before.

I am waiting for my time on home X-ray facility, and hope I can get
some suggestions before that.

Thanks,

Rongjin Guan

Re: [ccp4bb] cryo for crystals with CTAB as precipitant

[Vellieux Frédéric]

Hi there,

What about trying the ''olde'' method of a thin layer of oil around the 
crystal? No guarantee whatsoever that this will work but if you don't try it 
you won't know.

Fred.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Alejandro 
Madrigal Carrillo [amadri...@email.ifc.unam.mx]
Sent: Monday, February 22, 2016 11:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo for crystals with CTAB as precipitant

Dear All,


I got crystals using CTAB 10mM as precipitant, but I have had problems with the 
cryoprotectant solution which freezes.  Has anybody collected crystallographic 
data with crystals grown in this detergent? What cryoprotectant do you 
recommend to test?


Two papers report use of glycerol as cryoprotectant (Brown MA et al., 2005; 
glycerol 40% Ren A et al, 2010 ). But at -20oC the reservoir solution plus 
cryoprotectant freezes. I tried with many cryoprotectants  (glycerol 40%, EG 
35%, PEG 400 35%, hexanediol 50%, Sucrose 28%, Xylitol 30%, etc) but solution 
at -20oC looks cloudy or frozen.


Any suggestion will be appreciated.


Thanks in advance,

Alejandro Madrigal Carrillo, M.Sc.
Laboratorio de Biofísica de Biomacromoléculas, 205 sur
Departamento de Bioquímica y Biología Estructural
Instituto de Fisiología Celular
Universidad Nacional Autónoma de México
Circuito Exterior S/N. Ciudad Universitaria
México, D.F., México, 04510
Teléfono: +52 55 5622 5640
-

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Re: [ccp4bb] cryo-cooling, was: Re: [ccp4bb] crystallisation and mosaicity

[R.M. Garavito]

Jim,

The fact that liquid propane can exist at a range of temperatures is  
actually a MAJOR advantage.  While you must ensure that the  
temperature of the liquid propane is just above its own freezing  
point, the very high boiling point of propane ensures that there is  
liquid-to-solid contact with your sample for good heat transfer.   
When the cryogen boils, there is a gas-to-solid contact, slower  
cooling, and a greater chance of ice crystal formation.


Liquid-to-solid contact is important for getting the sample below  
~180K as fast as possible to get good sample freezing (i.e. glass  
formation), regardless if the sample is a crystal, an EM grid, or a  
small tissue block.  Thus, the jump from the temperature where a  
crystal/mother liquor freezes to the boiling point of the cryogen is  
a critical regime to pass through quickly.  That is why I prefer  
freezing in propane. But as you correctly point out, you must ensure  
that the temperature of the liquid propane is just its freezing point


Michael



R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  [EMAIL PROTECTED]



On Jun 6, 2008, at 1:09 PM, Jim Pflugrath wrote:

I would like to point out that flash-cooling in liquid propane has  
the added complication that the liquid propane can have a range of  
temperature and still be liquid.  If you use propane you may not  
know which temperature you are actually using.  The temperature in  
the exposed layer of the propane will be warmer (could be 231 K)  
than the bottom layer unless you stir the propane.  Luger's group  
has published their work on flash-cooling in propane.  [Raji, who  
posts here often must be away from the internet :) ] See  
Edayathumangalam and Luger (2005) Acta Cryst D 51, 891-898.


As an alternative to propane and ethane, one might consider carbon  
tetrafluoride instead.  It is a liquid between about 88 K and 145  
K, so not quite the range of propane, but if you see liquid, you  
know you are are below 145 K.  And it's non-flammable.  Note that  
the CRC has the wrong boiling point for this cryogenic gas which I  
believe is why CF4 is not used as much.


For more tips on cryo-cooling, see also the PDF linked at http:// 
www.rigaku.com/cryo/ and the references therein.


Jim

On Fri, 6 Jun 2008, R.M. Garavito wrote:


Tommi,

The question has been asked and answered not by protein  
crystallography, but by cyroelectron microscopy and EM freeze etch  
research.  Even as far back as the early 1960's, people noticed  
that liq. N2 was really slow at cooling. Read the cyroEM work on  
the bacteriorhodopsin photocycle and check out the wicked  
guillotine device for freezing.


The slower freezing in liq. N2 is partly due to nitrogen's low  
heat capacity, which can be seen in the fact that there is only  
about a 13 degree difference between the freezing and boiling  
points of N2 (~64K vs. ~77K).  In contrast, difference between the  
freezing and boiling points for propane is almost 148 degrees  
(~83K vs. ~231K).  Thus, it makes sense to freeze in liq. propane,  
but then shift to liq. N2 for storage and shipping.  Making  
propane popsicles for storage, shipping, and mounting is not  
necessary.


Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  [EMAIL PROTECTED]



On Jun 5, 2008, at 5:11 PM, Tommi Kajander wrote:


according to literature,see below and references
http://www.px.nsls.bnl.gov/courses/papers/ZD_EG_papers.html,
it is not clear that liq. propane plunged item would cool
faster. (whilst i havent tested this)...
Would anyone have actual experimental data with protein crystals
on the hyperquenching suggested by
Warkentin, V. Berejnov, and R. E. Thorne, J. Appl. Cryst. (2006) 39,
805-811. (no diffraction data in the paper). in particular with
small samples.
thanks,
Tommi
Quoting Petr Leiman <[EMAIL PROTECTED]>:
yes you are right, but I assumed if people see a cloud of  
condensed

fog over their LN2 bath they should remove that by
a) filling up the bowl completely e.g. some LN2 drips out of  
the bowl

b) blow the fog away before you dip
I think the original poster meant the relatively low heat  
conduction of
liquid N2, which causes boiling around the crystal immediately  
after

plunging.
The best way to freeze things is to put a small container of liquid
ethane
or propane into a liquid N2 bowl, and plunge into the ethane/ 
prop

Re: [ccp4bb] cryo-cooling, was: Re: [ccp4bb] crystallisation and mosaicity

[Jim Pflugrath]

Jim,

The fact that liquid propane can exist at a range of temperatures is actually 
a MAJOR advantage.  While you must ensure that the temperature of the liquid 
propane is just above its own freezing point, the very high boiling point of 
propane ensures that there is liquid-to-solid contact with your sample for 
good heat transfer.  When the cryogen boils, there is a gas-to-solid contact, 
slower cooling, and a greater chance of ice crystal formation.




Yes, I agree with the theory, but I also I believe that if you quickly 
plunge your crystal into liquid nitrogen, that the high velocity through 
the liquid presents fresh liquid nitrogen as you go and the gas-to-solid 
contact is neglible.  I think speed and having no cold-gas-layer above the 
liquid cryogen are the main points of the Warkentin et al. paper cited 
previously.  I have observed many slow people freezing crystals instead of 
flash-cooling them.


But for some crystals flash-cooling is better at temperatures higher than 
in the 77 K to 100 K regime for unknown reasons.  This can be one reason 
why flash-cooling in the gas stream occassionally is better.  It is also 
why liquid propane worked for Raji E. and Karolin L.: they were using a 
temperature above the freezing point of propane.


If you are having problems with flash-cooling, go ahead and try propane 
(my preference is CF4 over propane though).  And a trick we learned from 
the University of Cambridge is to fill a balloon with the gas, put the 
balloon opening over a 15 ml Falcon tube and then condense/freeze the gas 
in the tube for pouring into vials later.  This prevents wasting gas by 
bubbling through a coil held in liquid nitrogen.  This is just one of 
techniques described in the PDF I mentioned.


It is also not a bad idea to practice flash-cooling on lysozyme or 
thaumatin crystals and get good at it before working with your own 
precious crystals.


Jim