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

[Daniel M. Himmel, Ph. D.]

Dear JL,

Years ago, this was a common problem when I was crystallizing myosin
constructs for my doctoral work.  Some of the most beautiful crystals I
got showed little or no diffraction.  Often this occurs when there is a very
large water content in the asymmetric unit and in proteins that have a
great deal of intrinsic disorder.  Jon's suggestion to flash-cool them in
oil could work.  Your high PEG concentration in the flash-cooling solution
might work, too, but you probably cannot just plunge your crystals
suddenly into a much higher PEG solution.  A few suggestions:

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

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

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

I hope this helps.

-Daniel

On Tue, Aug 14, 2018 at 9:45 AM, ferrer  wrote:

> Hi
>
> Did you try them at room temp, in situ (straight in the plate). We observe
> that time to time on our beamline, when just harvesting, not mentioning
> cryo protection, is enough to loose all diffraction. It-s rare but happens.
>
> Regards
>
> JL
>
> On 14/08/2018 11:58, Careina Edgooms wrote:
>
> I got the most beautiful crystals I have ever seen and they don't diffract
> at all. Not poor diffraction, NO diffraction. Anyone know why this could be
> and how I can go about fixing it? I had three beautiful crystals and not
> one diffracted. I did leave them in the drop for about 3 weeks before
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> 
> Jean-Luc Ferrer
> Institut de Biologie Structurale71 Avenue des Martyrs 
> 
> CS 10090
> 38044 Grenoble Cedex 9 - FRANCE
>
> Ph.:  +33 (0)4 57 42 85 22
> Cell: +33 (0)6 89 45 13 57
> email: jean-luc.fer...@ibs.fr
> 
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[ferrer]

Dear Thomas,

Alternatively you can try shooting on crystals in the drop, in situ. So 
fishing, no cryo. But potentially high radiation damage. Can be 
considered if you have enough crystals, and if your crystallization 
plate makes it possible.


Regards

JL

On 14/08/2018 20:58, Thomas Krey wrote:


Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening 
vapor diffusion experiment in either


15% (v/v) Reagent alcohol

HEPES Na pH 7.5

0.2 M MgCl2

or in

27% Isopropanol

0.18 M MgCl2

90 mM HEPES Na pH 7.5

10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a 
good suggestion to stabilize the swirling movements? Does anyone have 
experience, whether these conditions alone can serve as 
cryo-protectant (i.e., do we really have to fish, move into cryo 
solution and fish again)?


Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas

Prof. Dr. Thomas Krey

Hannover Medical School

Institute of Virology

Structural Virology Group

Carl-Neuberg-Str. 1

D-30625 Hannover

phone: +49 (0) 511 - 532 4308

email: krey.tho...@mh-hannover.de




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



--

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

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





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

Re: [ccp4bb] Protein Purification- reg

[Kevin Jin]

Hi Amala,

1. Did you verify the sequence or the presence of the His-tag? If you were
not the person for cloning. I used to spend 14 months working on an clone
and eventually I was allowed to check the sequence and verify the
expression of His-tag by Western-Blot. There was no his-tag.
2. You can try ionic-exchange columns instead of Ni-Column.  In this case,
chaining the cationic column and anionic column in a linear way. You target
should either stay in 1) cation or 2) anionic or 3) flow through. You can
verify the presence by SDS page.
3. What's the temperature you used for purification? I.e. if the pH 7.5 of
tris was under RT, then it should be 8.3 in cold room.
4. Increasing the concentration of Na+ will increase protein solubility and
decrease non-specific binding ( possible langmuir adsoprtion). Normally,
people use 200-300 mM NaCl. If the Tris-tri sodium is used, the
concentration may need to controlled < 50mM. For instance, if 50mM Tris-tri
Na was used, then 150mM NaCl could be introduced in the buffer system
additionally.
5. 10mM Imidazole is used for removing non-specific adsorption in this
case.
6. In some cases, everything looks normal, but the protein just does like
to bind to the Ni(Co)- column. In this case, using ionic-columns will be an
alternative method before you try another vector or expression system.

The advantage of ionic-columns is you can buy the beads (+ and -) from
sigma with much less cost. You even don't need to use an AKTA system.

I hope this will be helpful and see your publication soon.

Kevin



On Mon, Aug 13, 2018 at 6:50 AM amala mathimaran 
wrote:

> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa histidine). The
> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
> *E.coli
> *BL21 cells. The expression was good. I am trying to purify a protein
> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
> as buffer A but 250mM imidazole). I eluted the protein in step wise
> gradiant. the protein was less binded in Ni affinity IMAC column because
> eluted fraction contain less amount and the protein remain present in the
> Flow through. Can any one suggest how to increase the binding affinity of
> the protein and how to purify the protein. The protein PI was 6.33
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Kevin Jin]

I just give an example of oil. Indeed, paraffin is also a very good option.
In some cases (<=5% alcohol).  Here is the difference I observed between
paraffin and paratone oil.
1. Paraffin oil has low viscosity. Paratone oil is too sticky with high
viscosity. Sometimes, I mix them with different ratio.
2. In the crystallization with high concentration of alcohol (>10%), I
prefer paratone oil, which can form a bulky cover immediately. Paraffin oil
may spread out and could not cover the drop completely.

The basic idea is to proved an effective shell to prevent solvent
evaporation.



On Tue, Aug 14, 2018 at 12:56 PM Patrick Loll  wrote:

> I second (third?) what Tommi and Kevin said about using an oil to cover
> the drop to slow evaporation (I like paraffin for this—not too viscous).
> Here’s an additional nuance: Saturate the oil with the alcohol first,
> before using it to cover the drop.
>
> > On 14 Aug 2018, at 2:58 PM, Thomas Krey 
> wrote:
> >
> > Dear crystallization experts,
> >
> > We have 3D protein crystals grown from a microseed matrix screening
> vapor diffusion experiment in either
> >
> > 15% (v/v) Reagent alcohol
> > HEPES Na pH 7.5
> > 0.2 M MgCl2
> >
> > or in
> >
> > 27% Isopropanol
> > 0.18 M MgCl2
> > 90 mM HEPES Na pH 7.5
> > 10% Glycerol
> >
> > Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
> > Any suggestion or input would be highly welcome.
> >
> > Thank you very much in advance.
> >
> > Thomas
> >
> >
> > Prof. Dr. Thomas Krey
> > Hannover Medical School
> > Institute of Virology
> > Structural Virology Group
> > Carl-Neuberg-Str. 1
> > D-30625 Hannover
> > phone: +49 (0) 511 - 532 4308
> > email: krey.tho...@mh-hannover.de
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
>
> ---
> Patrick J. Loll, Ph. D.
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Patrick Loll]

I second (third?) what Tommi and Kevin said about using an oil to cover the 
drop to slow evaporation (I like paraffin for this—not too viscous). Here’s an 
additional nuance: Saturate the oil with the alcohol first, before using it to 
cover the drop. 

> On 14 Aug 2018, at 2:58 PM, Thomas Krey  wrote:
> 
> Dear crystallization experts,
>  
> We have 3D protein crystals grown from a microseed matrix screening vapor 
> diffusion experiment in either
>  
> 15% (v/v) Reagent alcohol
> HEPES Na pH 7.5
> 0.2 M MgCl2 
>  
> or in 
>  
> 27% Isopropanol
> 0.18 M MgCl2
> 90 mM HEPES Na pH 7.5
> 10% Glycerol
>  
> Upon opening the corresponding wells these crystals move quite a bit – 
> presumably due to the volatility of the alcohols. Does anyone have a good 
> suggestion to stabilize the swirling movements? Does anyone have experience, 
> whether these conditions alone can serve as cryo-protectant (i.e., do we 
> really have to fish, move into cryo solution and fish again)? 
> Any suggestion or input would be highly welcome.
>  
> Thank you very much in advance.
>  
> Thomas
>  
>  
> Prof. Dr. Thomas Krey
> Hannover Medical School  
> Institute of Virology
> Structural Virology Group
> Carl-Neuberg-Str. 1 
> D-30625 Hannover
> phone: +49 (0) 511 - 532 4308
> email: krey.tho...@mh-hannover.de
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Kevin Jin]

Hi Thomas,

This is usual when high volatile solvent is used in crystallization
(membrane or glycoproteins). The crystal may looks very nice with sharp
edges. When you open the cover glass, you may see a very thin film formed
on the hang-on drops. Once you touch the drop, then crystals move very
quickly like flying and start cracking.

Here is what you may try:
1. When you open the cover glass, using a big drop of Paratone oil to cover
(wrap) the drop immediately and completely.
2. Then, inject the cryoprotectant into the drop wrapped by paratone oil.
3.  When you wash the crystal in cryoprotectant and fish the crystal out,
please always keep all of the operation in a large paratone oil drop.
4. Make sure there is always a layer of paratone oil on the loop when you
freeze it in LN2.

I hope this will be helpful.

Regards,

Kevin





On Tue, Aug 14, 2018 at 12:00 PM Thomas Krey 
wrote:

> Dear crystallization experts,
>
>
>
> We have 3D protein crystals grown from a microseed matrix screening vapor
> diffusion experiment in either
>
>
>
> 15% (v/v) Reagent alcohol
>
> HEPES Na pH 7.5
>
> 0.2 M MgCl2
>
>
>
> or in
>
>
>
> 27% Isopropanol
>
> 0.18 M MgCl2
>
> 90 mM HEPES Na pH 7.5
>
> 10% Glycerol
>
>
>
> Upon opening the corresponding wells these crystals move quite a bit –
> presumably due to the volatility of the alcohols. Does anyone have a good
> suggestion to stabilize the swirling movements? Does anyone have
> experience, whether these conditions alone can serve as cryo-protectant
> (i.e., do we really have to fish, move into cryo solution and fish again)?
>
> Any suggestion or input would be highly welcome.
>
>
>
> Thank you very much in advance.
>
>
>
> Thomas
>
>
>
>
>
> Prof. Dr. Thomas Krey
>
> Hannover Medical School
>
> Institute of Virology
>
> Structural Virology Group
>
> Carl-Neuberg-Str. 1
>
> D-30625 Hannover
>
> phone: +49 (0) 511 - 532 4308
>
> email: krey.tho...@mh-hannover.de
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Petri Kursula]

Hi,
you could try picking in the cold room. Provided the temperature change does 
not kill the crystals, this sometimes worked fine for me in similar cases.
Petri


Petri Kursula
--
Professor 
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula 

petri.kurs...@uib.no 
--
Group Leader, Adjunct Professor
Faculty of Biochemistry and Molecular Medicine
University of Oulu, Finland
--



> On 14 Aug 2018, at 20:58, Thomas Krey  wrote:
> 
> Dear crystallization experts,
>  
> We have 3D protein crystals grown from a microseed matrix screening vapor 
> diffusion experiment in either
>  
> 15% (v/v) Reagent alcohol
> HEPES Na pH 7.5
> 0.2 M MgCl2 
>  
> or in 
>  
> 27% Isopropanol
> 0.18 M MgCl2
> 90 mM HEPES Na pH 7.5
> 10% Glycerol
>  
> Upon opening the corresponding wells these crystals move quite a bit – 
> presumably due to the volatility of the alcohols. Does anyone have a good 
> suggestion to stabilize the swirling movements? Does anyone have experience, 
> whether these conditions alone can serve as cryo-protectant (i.e., do we 
> really have to fish, move into cryo solution and fish again)? 
> Any suggestion or input would be highly welcome.
>  
> Thank you very much in advance.
>  
> Thomas
>  
>  
> Prof. Dr. Thomas Krey
> Hannover Medical School  
> Institute of Virology
> Structural Virology Group
> Carl-Neuberg-Str. 1 
> D-30625 Hannover
> phone: +49 (0) 511 - 532 4308
> email: krey.tho...@mh-hannover.de 
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Kajander, Tommi A]

Hi, One way is to cover the drop with e.g. paraffin oil (to prevent 
evaporation) and fish it out in the oil. Have had luck with that in some cases.

Tommi

On 14 Aug 2018, at 22:09, Bonsor, Daniel 
mailto:dbon...@som.umaryland.edu>> wrote:

I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir 
to the drop. The larger volume of the drop appears to slow down the whizzing of 
the crystals and allows you to get a few crystals. Though it still occurs. You 
could also cool the area down or move into the cold room if your crystals 
survive the transfer as evaporation should be less at 4oC.

You can also spot several 1-2uL of neat reservoir solution around the drop to 
create a local vapor barrier to prevent evaporation of the drop. It can 
completely stop the movement of crystals for a few minutes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas 
Krey
Sent: Tuesday, August 14, 2018 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fishing crystals from volatile solvent as precipitant

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email 
system. Whether the sender is known or not known, hover over any links before 
clicking and use caution opening attachments.

Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de




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



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



Tommi Kajander, Ph.D., PI
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1 (P.O. Box 65)
00014 Helsinki, Finland
p. +358-2-941-58904 / +358-050-4480991
tommi.kajan...@helsinki.fi
http://www.biocenter.helsinki.fi/bi/kajander/






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

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Bonsor, Daniel]

I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir 
to the drop. The larger volume of the drop appears to slow down the whizzing of 
the crystals and allows you to get a few crystals. Though it still occurs. You 
could also cool the area down or move into the cold room if your crystals 
survive the transfer as evaporation should be less at 4oC.

You can also spot several 1-2uL of neat reservoir solution around the drop to 
create a local vapor barrier to prevent evaporation of the drop. It can 
completely stop the movement of crystals for a few minutes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas 
Krey
Sent: Tuesday, August 14, 2018 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fishing crystals from volatile solvent as precipitant

CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email 
system. Whether the sender is known or not known, hover over any links before 
clicking and use caution opening attachments.

Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de




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



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

[ccp4bb] Fishing crystals from volatile solvent as precipitant

[Thomas Krey]

Dear crystallization experts,

We have 3D protein crystals grown from a microseed matrix screening vapor 
diffusion experiment in either

15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2

or in

27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol

Upon opening the corresponding wells these crystals move quite a bit – 
presumably due to the volatility of the alcohols. Does anyone have a good 
suggestion to stabilize the swirling movements? Does anyone have experience, 
whether these conditions alone can serve as cryo-protectant (i.e., do we really 
have to fish, move into cryo solution and fish again)?
Any suggestion or input would be highly welcome.

Thank you very much in advance.

Thomas


Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de




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

Re: [ccp4bb] Protein Purification- reg

[Rajnandani Kashyap]

Dear Amala
Please increase NaCl concentration to 200mM from 50mM. That can help you
out by increasing affinity of your protein to bead and will delay the
elution time.


On Mon, Aug 13, 2018, 7:20 PM amala mathimaran  wrote:

> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa histidine). The
> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
> *E.coli
> *BL21 cells. The expression was good. I am trying to purify a protein
> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
> as buffer A but 250mM imidazole). I eluted the protein in step wise
> gradiant. the protein was less binded in Ni affinity IMAC column because
> eluted fraction contain less amount and the protein remain present in the
> Flow through. Can any one suggest how to increase the binding affinity of
> the protein and how to purify the protein. The protein PI was 6.33
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

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

[ferrer]

On 14/08/2018 12:27, Careina Edgooms wrote:
Cryoprotectant was 50% PEG just added to the buffer that it 
crystallised in. It crystallised under batch.

which kind of batch plate ? Some are not suitable to in situ.

JL


On Tuesday, August 14, 2018, 12:11:17 PM GMT+2, Hughes, Jon 
 wrote:



maybe it's the cryobuffer that's the problem (you didn't mention it). 
you could try to fish the crystals with minimal liquid attached by 
mounting them in oil rather than a cryobuffer. or you could test the 
native diffraction "in situ" (at room temperature in the drop): quite 
a few beamlines offer this possibility these days.


best

jon

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *Careina Edgooms

*Gesendet:* Dienstag, 14. August 2018 11:59
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] crystals that dont diffract :( :(

I got the most beautiful crystals I have ever seen and they don't 
diffract at all. Not poor diffraction, NO diffraction. Anyone know why 
this could be and how I can go about fixing it? I had three beautiful 
crystals and not one diffracted. I did leave them in the drop for 
about 3 weeks before harvesting and in liquid nitrogen for about a 
month before diffracting. Could that be a factor? If I regrew more 
beautiful crystals and diffracted straight away could that help?


Careina



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




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




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



--

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

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





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

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

[ferrer]

Hi

Did you try them at room temp, in situ (straight in the plate). We 
observe that time to time on our beamline, when just harvesting, not 
mentioning cryo protection, is enough to loose all diffraction. It-s 
rare but happens.


Regards

JL

On 14/08/2018 11:58, Careina Edgooms wrote:
I got the most beautiful crystals I have ever seen and they don't 
diffract at all. Not poor diffraction, NO diffraction. Anyone know why 
this could be and how I can go about fixing it? I had three beautiful 
crystals and not one diffracted. I did leave them in the drop for 
about 3 weeks before harvesting and in liquid nitrogen for about a 
month before diffracting. Could that be a factor? If I regrew more 
beautiful crystals and diffracted straight away could that help?

Careina



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



--

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

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





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

Re: [ccp4bb] Protein Purification- reg

[Manjula Ramu]

Hello Amala,

Usually Ni-NTA won't have this kind of problem of binding. Probably your
protein is have interaction with hexagon his tag which is affecting its
affinity towards beads. You can try putting tag in C- terminal end of the
protein.


On Mon, 13 Aug 2018, 8:02 pm Artem Evdokimov, 
wrote:

> Hi Amala,
>
> Depending on the resin you used there may be a conflict with BME and also
> 50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5
> and TCEP instead of BME, or use 30 mM TRIS at pH 8.0
>
> Alternatively your protein is aggregated and does not bind well...
>
> Artem
>
> - Cosmic Cats approve of this message
>
> On Mon, Aug 13, 2018 at 9:49 AM, amala mathimaran 
> wrote:
>
>> Dear All
>>
>> I am working with HIS – tag protein in N-terminal (hexa histidine). The
>> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
>> *E.coli
>> *BL21 cells. The expression was good. I am trying to purify a protein
>> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
>> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
>> as buffer A but 250mM imidazole). I eluted the protein in step wise
>> gradiant. the protein was less binded in Ni affinity IMAC column because
>> eluted fraction contain less amount and the protein remain present in the
>> Flow through. Can any one suggest how to increase the binding affinity of
>> the protein and how to purify the protein. The protein PI was 6.33
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] crystals that don’t diffract :( :(

[Fischmann, Thierry]

It does happen that “beautiful” crystals have no diffraction. Here is an 
example :
https://www.sciencedirect.com/science/article/pii/0022283691906078

In my experience, crystals are fairly stable. But not always: crystals of human 
endothelial nitric oxide synthase or inducible nitric oxide synthase catalytic 
domain lasted two weeks at most before losing their diffraction completely.

The method used to freeze the crystal can be a factor. It is worth checking at 
room temperature.

But your crystals are not necessarily a complete loss: they can be used as 
micro-seeds added to a broad screen, and help discover new crystal forms.

Thierry


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina 
Edgooms
Sent: Tuesday, August 14, 2018 5:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystals that dont diffract :( :(

EXTERNAL EMAIL – Use caution with any links or file attachments.
I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Notice:  This e-mail message, together with any attachments, contains
information of Merck & Co., Inc. (2000 Galloping Hill Road, Kenilworth,
New Jersey, USA 07033), and/or its affiliates Direct contact information
for affiliates is available at 
http://www.merck.com/contact/contacts.html) that may be confidential,
proprietary copyrighted and/or legally privileged. It is intended solely
for the use of the individual or entity named on this message. If you are
not the intended recipient, and have received this message in error,
please notify us immediately by reply e-mail and then delete it from 
your system.




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

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

[zaigham mahmood khan]

Hey Careina

I had once experience the same phenomenon. No diffraction at all. That did
not make any sense to me. So i dipped the "empty" loop in the water, and
flash froze the loop in liquid nitrogen. Once exposed to X-ray, there was
no ice rings... Eventually, it turned out that system had a glitch, and
there was no X-ray. True story!

Best wishes

-Z


Zaigham Mahmood Khan, PhD

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

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

> I got the most beautiful crystals I have ever seen and they don't diffract
> at all. Not poor diffraction, NO diffraction. Anyone know why this could be
> and how I can go about fixing it? I had three beautiful crystals and not
> one diffracted. I did leave them in the drop for about 3 weeks before
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

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

[Rangana Warshamanage]

Hi Careina,

Some other things to check for:

Where did you measure the crystal, on a home source or at a synchrotron? if
at a synchrotron, try to put bit more flux and see whether you see any
diffraction?
Also check the beam and your crystal alignment.

Rangana


On Tue, Aug 14, 2018 at 12:15 PM, Andreas Forster 
wrote:

> Hi Careina,
>
> if you don't have grey hair (available in the lab), you can still mount
> crystals at room temperature.  With a MiTeGen RT kit, very little skill is
> required to test crystal diffraction or even collect entire data sets at
> room temperature.
> https://www.mitegen.com/product/micrort-room-temperature-starter-kits/
>
> All best.
>
>
> Andreas
>
>
>
> On Tue, Aug 14, 2018 at 12:48 PM, Harry Powell <193323b1e616-dmarc-
> requ...@jiscmail.ac.uk> wrote:
>
>> Hi
>>
>> One other thing to try that someone with grey hair in your lab might know
>> about - mount a "crystal" in a capillary and see if it diffracts at room
>> temp. As long as you have a source and detector in your home lab, there's
>> no need to go to a synchrotron and use their in situ facilities.
>>
>> There are those of us who would contend that if your sample doesn't
>> diffract, then it isn't a crystal, no matter how nice it looks (though it
>> might have been one once...)!
>>
>> Harry
>> --
>> Dr Harry Powell
>> Chairman of European Crystallographic Association SIG9 (Crystallographic
>> Computing)
>>
>>
>>
>>
>> On 14 Aug 2018, at 11:14, Elspeth Garman wrote:
>>
>> Yes, essential to test at room temperature without changing their buffer
>> medium before getting worried!
>> If they don’t diffract at RT, they are very very unlikely to diffract at
>> cryotemperatures, whatever you do to them before hand!
>> Best wishes
>> Elspeth
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
>> Of *Hughes, Jon
>> *Sent:* 14 August 2018 11:11
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>>
>> maybe it's the cryobuffer that's the problem (you didn't mention it). you
>> could try to fish the crystals with minimal liquid attached by mounting
>> them in oil rather than a cryobuffer. or you could test the native
>> diffraction "in situ" (at room temperature in the drop): quite a few
>> beamlines offer this possibility these days.
>> best
>> jon
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
>> ] *Im Auftrag von *Careina Edgooms
>> *Gesendet:* Dienstag, 14. August 2018 11:59
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [ccp4bb] crystals that dont diffract :( :(
>>
>> I got the most beautiful crystals I have ever seen and they don't
>> diffract at all. Not poor diffraction, NO diffraction. Anyone know why this
>> could be and how I can go about fixing it? I had three beautiful crystals
>> and not one diffracted. I did leave them in the drop for about 3 weeks
>> before harvesting and in liquid nitrogen for about a month before
>> diffracting. Could that be a factor? If I regrew more beautiful crystals
>> and diffracted straight away could that help?
>> Careina
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

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

[Andreas Forster]

Hi Careina,

if you don't have grey hair (available in the lab), you can still mount
crystals at room temperature.  With a MiTeGen RT kit, very little skill is
required to test crystal diffraction or even collect entire data sets at
room temperature.
https://www.mitegen.com/product/micrort-room-temperature-starter-kits/

All best.


Andreas



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

> Hi
>
> One other thing to try that someone with grey hair in your lab might know
> about - mount a "crystal" in a capillary and see if it diffracts at room
> temp. As long as you have a source and detector in your home lab, there's
> no need to go to a synchrotron and use their in situ facilities.
>
> There are those of us who would contend that if your sample doesn't
> diffract, then it isn't a crystal, no matter how nice it looks (though it
> might have been one once...)!
>
> Harry
> --
> Dr Harry Powell
> Chairman of European Crystallographic Association SIG9 (Crystallographic
> Computing)
>
>
>
>
> On 14 Aug 2018, at 11:14, Elspeth Garman wrote:
>
> Yes, essential to test at room temperature without changing their buffer
> medium before getting worried!
> If they don’t diffract at RT, they are very very unlikely to diffract at
> cryotemperatures, whatever you do to them before hand!
> Best wishes
> Elspeth
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Hughes,
> Jon
> *Sent:* 14 August 2018 11:11
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>
> maybe it's the cryobuffer that's the problem (you didn't mention it). you
> could try to fish the crystals with minimal liquid attached by mounting
> them in oil rather than a cryobuffer. or you could test the native
> diffraction "in situ" (at room temperature in the drop): quite a few
> beamlines offer this possibility these days.
> best
> jon
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
> ] *Im Auftrag von *Careina Edgooms
> *Gesendet:* Dienstag, 14. August 2018 11:59
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [ccp4bb] crystals that dont diffract :( :(
>
> I got the most beautiful crystals I have ever seen and they don't diffract
> at all. Not poor diffraction, NO diffraction. Anyone know why this could be
> and how I can go about fixing it? I had three beautiful crystals and not
> one diffracted. I did leave them in the drop for about 3 weeks before
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

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

[Tom Peat]

?We have some crystals that diffract well when fresh (less than one week old) 
but lose almost all diffraction by the end of 2 weeks, so age can matter. One 
crystals are in liquid nitrogen, they should be safe from further degradation, 
but may suffer from ice contamination.

cheers, tom


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

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

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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



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

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

[Harry Powell]

Hi

One other thing to try that someone with grey hair in your lab might know about 
- mount a "crystal" in a capillary and see if it diffracts at room temp. As 
long as you have a source and detector in your home lab, there's no need to go 
to a synchrotron and use their in situ facilities.

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

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




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

> Yes, essential to test at room temperature without changing their buffer 
> medium before getting worried!
> If they don’t diffract at RT, they are very very unlikely to diffract at 
> cryotemperatures, whatever you do to them before hand!
> Best wishes
> Elspeth
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
> Jon
> Sent: 14 August 2018 11:11
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(
>  
> maybe it's the cryobuffer that's the problem (you didn't mention it). you 
> could try to fish the crystals with minimal liquid attached by mounting them 
> in oil rather than a cryobuffer. or you could test the native diffraction "in 
> situ" (at room temperature in the drop): quite a few beamlines offer this 
> possibility these days.
> best
> jon
>  
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Careina Edgooms
> Gesendet: Dienstag, 14. August 2018 11:59
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [ccp4bb] crystals that dont diffract :( :(
>  
> I got the most beautiful crystals I have ever seen and they don't diffract at 
> all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
> how I can go about fixing it? I had three beautiful crystals and not one 
> diffracted. I did leave them in the drop for about 3 weeks before harvesting 
> and in liquid nitrogen for about a month before diffracting. Could that be a 
> factor? If I regrew more beautiful crystals and diffracted straight away 
> could that help?
> Careina
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 




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

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

[fourati zaineb]

 
Dear Careina,




Unfortunately there is no direct connection between nice looking crystals and 
good diffraction.

Sometimes ugly crystals can diffract well while nice ones don’t. 

You may try crystal dehydration ; it allows to remove excess of solvent and 
change crystal packing which can lead to better crystal order and better 
diffraction.

Here are some examples described in the literature




https://www.ncbi.nlm.nih.gov/pubmed/24311593

https://www.ncbi.nlm.nih.gov/pubmed/22232185

https://www.ncbi.nlm.nih.gov/pubmed/12575933




Bests,




Zaineb

Le mardi 14 août 2018 à 12:11:23 UTC+2, Hughes, Jon 
 a écrit :  
 
 #yiv5746868635 #yiv5746868635 -- _filtered #yiv5746868635 
{font-family:Helvetica;panose-1:2 11 6 4 2 2 2 2 2 4;} _filtered #yiv5746868635 
{panose-1:2 4 5 3 5 4 6 3 2 4;} _filtered #yiv5746868635 
{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;}#yiv5746868635 
#yiv5746868635 p.yiv5746868635MsoNormal, #yiv5746868635 
li.yiv5746868635MsoNormal, #yiv5746868635 div.yiv5746868635MsoNormal 
{margin:0cm;margin-bottom:.0001pt;font-size:12.0pt;font-family:New 
serif;}#yiv5746868635 a:link, #yiv5746868635 span.yiv5746868635MsoHyperlink 
{color:blue;text-decoration:underline;}#yiv5746868635 a:visited, #yiv5746868635 
span.yiv5746868635MsoHyperlinkFollowed 
{color:purple;text-decoration:underline;}#yiv5746868635 p 
{margin-right:0cm;margin-left:0cm;font-size:12.0pt;font-family:New 
serif;}#yiv5746868635 p.yiv5746868635msonormal0, #yiv5746868635 
li.yiv5746868635msonormal0, #yiv5746868635 div.yiv5746868635msonormal0 
{margin-right:0cm;margin-left:0cm;font-size:12.0pt;font-family:New 
serif;}#yiv5746868635 span.yiv5746868635E-MailFormatvorlage19 
{font-family:sans-serif;color:#2E74B5;}#yiv5746868635 
.yiv5746868635MsoChpDefault {font-size:10.0pt;} _filtered #yiv5746868635 
{margin:70.85pt 70.85pt 2.0cm 70.85pt;}#yiv5746868635 
div.yiv5746868635WordSection1 {}#yiv5746868635 
maybe it's the cryobuffer that's the problem (you didn't mention it). you could 
try to fish the crystals with minimal liquid attached by mounting them in oil 
rather than a cryobuffer. or you could test the native diffraction "in situ" 
(at room temperature in the drop): quite a few beamlines offer this possibility 
these days.
 
best
 
jon
 
  
 
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK]Im Auftrag von Careina 
Edgooms
Gesendet: Dienstag, 14. August 2018 11:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] crystals that dont diffract :( :(
 
  
 
I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
 
Careina
 
  
 
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
 

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



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

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

[Tim Gruene]

Dear Careina,

you could use the old crystals, that did not diffract, for microseeding
to regrew nicer crystals. Once you have them, try to use them as quickly
as possible. Three weeks can be a long time for crystals.

Storage in liquid nitrogen should not be the problem.

Best,
Tim


On 08/14/2018 11:58 AM, Careina Edgooms wrote:
> I got the most beautiful crystals I have ever seen and they don't
> diffract at all. Not poor diffraction, NO diffraction. Anyone know why
> this could be and how I can go about fixing it? I had three beautiful
> crystals and not one diffracted. I did leave them in the drop for about
> 3 weeks before harvesting and in liquid nitrogen for about a month
> before diffracting. Could that be a factor? If I regrew more beautiful
> crystals and diffracted straight away could that help?
> Careina
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

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

GPG Key ID = A46BEE1A




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


signature.asc
Description: OpenPGP digital signature

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

[Careina Edgooms]

 Cryoprotectant was 50% PEG just added to the buffer that it crystallised in. 
It crystallised under batch.
On Tuesday, August 14, 2018, 12:11:17 PM GMT+2, Hughes, Jon 
 wrote:  
 
 #yiv2688348338 #yiv2688348338 -- _filtered #yiv2688348338 
{font-family:Helvetica;panose-1:2 11 6 4 2 2 2 2 2 4;} _filtered #yiv2688348338 
{panose-1:2 4 5 3 5 4 6 3 2 4;} _filtered #yiv2688348338 
{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;}#yiv2688348338 
#yiv2688348338 p.yiv2688348338MsoNormal, #yiv2688348338 
li.yiv2688348338MsoNormal, #yiv2688348338 div.yiv2688348338MsoNormal 
{margin:0cm;margin-bottom:.0001pt;font-size:12.0pt;font-family:New 
serif;}#yiv2688348338 a:link, #yiv2688348338 span.yiv2688348338MsoHyperlink 
{color:blue;text-decoration:underline;}#yiv2688348338 a:visited, #yiv2688348338 
span.yiv2688348338MsoHyperlinkFollowed 
{color:purple;text-decoration:underline;}#yiv2688348338 p 
{margin-right:0cm;margin-left:0cm;font-size:12.0pt;font-family:New 
serif;}#yiv2688348338 p.yiv2688348338msonormal0, #yiv2688348338 
li.yiv2688348338msonormal0, #yiv2688348338 div.yiv2688348338msonormal0 
{margin-right:0cm;margin-left:0cm;font-size:12.0pt;font-family:New 
serif;}#yiv2688348338 span.yiv2688348338E-MailFormatvorlage19 
{font-family:sans-serif;color:#2E74B5;}#yiv2688348338 
.yiv2688348338MsoChpDefault {font-size:10.0pt;} _filtered #yiv2688348338 
{margin:70.85pt 70.85pt 2.0cm 70.85pt;}#yiv2688348338 
div.yiv2688348338WordSection1 {}#yiv2688348338 
maybe it's the cryobuffer that's the problem (you didn't mention it). you could 
try to fish the crystals with minimal liquid attached by mounting them in oil 
rather than a cryobuffer. or you could test the native diffraction "in situ" 
(at room temperature in the drop): quite a few beamlines offer this possibility 
these days.
 
best
 
jon
 
  
 
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK]Im Auftrag von Careina 
Edgooms
Gesendet: Dienstag, 14. August 2018 11:59
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] crystals that dont diffract :( :(
 
  
 
I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
 
Careina
 
  
 
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
 

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



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

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

[Elspeth Garman]

Yes, essential to test at room temperature without changing their buffer medium 
before getting worried!
If they don’t diffract at RT, they are very very unlikely to diffract at 
cryotemperatures, whatever you do to them before hand!
Best wishes
Elspeth

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: 14 August 2018 11:11
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] crystals that dont diffract :( :(

maybe it's the cryobuffer that's the problem (you didn't mention it). you could 
try to fish the crystals with minimal liquid attached by mounting them in oil 
rather than a cryobuffer. or you could test the native diffraction "in situ" 
(at room temperature in the drop): quite a few beamlines offer this possibility 
these days.
best
jon

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

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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



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



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

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

[Hughes, Jon]

maybe it's the cryobuffer that's the problem (you didn't mention it). you could 
try to fish the crystals with minimal liquid attached by mounting them in oil 
rather than a cryobuffer. or you could test the native diffraction "in situ" 
(at room temperature in the drop): quite a few beamlines offer this possibility 
these days.
best
jon

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

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?
Careina



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



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

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

[Nikolas]

Hello Careina,

Please send pics or didn't happen.
Anyway, in my very short experience, ugly crystals can diffract better than
beautiful ones. But have you  checked first if they were  protein crystals
or not?

Best of luck,
Nikk

On Tue, 14 Aug 2018, 11:59 Careina Edgooms, <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> I got the most beautiful crystals I have ever seen and they don't diffract
> at all. Not poor diffraction, NO diffraction. Anyone know why this could be
> and how I can go about fixing it? I had three beautiful crystals and not
> one diffracted. I did leave them in the drop for about 3 weeks before
> harvesting and in liquid nitrogen for about a month before diffracting.
> Could that be a factor? If I regrew more beautiful crystals and diffracted
> straight away could that help?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

[ccp4bb] crystals that dont diffract :( :(

[Careina Edgooms]

I got the most beautiful crystals I have ever seen and they don't diffract at 
all. Not poor diffraction, NO diffraction. Anyone know why this could be and 
how I can go about fixing it? I had three beautiful crystals and not one 
diffracted. I did leave them in the drop for about 3 weeks before harvesting 
and in liquid nitrogen for about a month before diffracting. Could that be a 
factor? If I regrew more beautiful crystals and diffracted straight away could 
that help?Careina



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

[ccp4bb] SPring-8/CCP4 workshop on data collection and structure solution

[Charles Ballard - UKRI STFC]

Dear All

10 days to the registration closure for the second CCP4/Spring-8 (RIKEN) 
workshop running from 1-6 October 2018.

This workshop will cover data collection, processing and structure solution.  
Details are available at
http://www.ccp4.ac.uk/schools/Japan-2018 , with more to follow.  Registration 
is on the same site.  Registration will
close on 24 August.

Topics will include XDS (Kay Diederichs), DIALS (Graeme Winter), the CCP4 suite 
(myself and others),
PDB_REDO (Robbie Joosten), COOT and others. The format will cover data 
collection, lectures, tutorials and
problem solving, so students are encourages to bring their own data to work on.

Charles and Kunio.



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

[ccp4bb] Postdoctoral position in cryo-EM at NNF-CPR, University of Copenhagen

[Nicholas M I Taylor]

Dear all,

I would like to bring to your attention a postdoctoral position in my research 
group at NNF-CPR, University of Copenhagen, to study the structure and function 
of contractile injection systems targeting eukaryotic cells. The position is 
fully funded and is for 2 years in the first instance, extendable to 4 years. 
Recent relevant publications include Taylor et al., Nature, 2016 (PMID 
27193680) and Taylor et al., Mol. 
Microbiol., 2018 (PMID 29405518). 
We have excellent access to state-of-the-art cryo-EM facilities including a 
Titan Krios with Falcon 3EC direct electron detector and Volta phase plates.

Applications need to be sent through the online application system, accessible 
here:
https://employment.ku.dk/faculty/?show=147807

More information about the group can be found here:
https://www.cpr.ku.dk/research/protein-structure-function-program/taylor/

Informal enquiries can be directed to 
nicholas.tay...@cpr.ku.dk.

Best wishes,

Nicholas

Nicholas M. I. Taylor, PhD
Associate Professor

University of Copenhagen
Faculty of Health and Medical Sciences
Novo Nordisk Foundation Center for Protein Research
Blegdamsvej 3B, building 6.1
2200 Copenhagen N
Denmark

TEL +45 35 33 53 37
nicholas.tay...@cpr.ku.dk
http://www.cpr.ku.dk/research/protein-structure-function-program/taylor

[cid:image001.gif@01D3ADA6.1B29D8A0] [cid:image002.png@01D3ADA6.1B29D8A0] 





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