Re: [ccp4bb] Protein or DNA crystals

[Patrick Shaw Stewart]

We have sometimes used Michael Garavito's excellent suggestion of diffusing
glutaraldehyde into the drops - mentioned on this board a few days ago.
Our protein crystals either turned brown or turned into brown goo.

Michael and others, what effect would you expect glutaraldehyde to have on
DNA crystals?

Patrick


On 19 June 2017 at 15:20, Joseph Ho  wrote:

> Dear all:
>
> I would like to seek your opinion on our crystal hits. We are working
> on protein/dsDNA complex. By changing different protein and DNA
> (14-22bp) constructs, we recently got some hits from commercial
> screens using sitting drop vapor diffusion (very small xtals). The
> precipitant is PEG and the picture of crystals are attached. In this
> particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
> 100mM NaCl. The crystal seems floating and sit in the bottom. We do
> some test shot from other conditions and it is not salt crystals. The
> crystals can suck in izit dye.  I do some google and it seems izit dye
> also turns dsDNA crystal into blue. We also do UV/Vis microscope but
> no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.
>
> This is our first time to work on protein/DNA complex crystals and we
> are not certain if this is just DNA or protein/DNA crystals. Can you
> provide your comments on our hits?
>
> Thank you for your help
>
> Joseph
>



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

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] Protein or DNA crystals

[Diana Tomchick]

It’s been my experience that protein crystals with that high a percentage of 
tryptophan residues (>2%) should give a very clearly positive result from a UV 
microscope such as a Jansi UVEX.

Diana

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

On Jun 19, 2017, at 9:20 AM, Joseph Ho  wrote:

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph





UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] Protein or DNA crystals

[Phoebe A. Rice]

This is not at all conclusive, but one depressing thing is that DNA-only 
crystals are often hexagonal (like yours are).  
Can you reproduce them without the protein? 

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
pr...@uchicago.edu



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Joseph Ho 
[sbddintai...@gmail.com]
Sent: Monday, June 19, 2017 9:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein or DNA crystals

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph

Re: [ccp4bb] Protein or DNA crystals

[CHAVAS Leonard]

Dear Kumar

you said 'shot from other conditions': we do not know then if you shot these 
crystals or not.

Else: 
- DNA should give you an X pattern, most of the time.
- protein should give you at least low resolution pattern, or faint rings if it 
is powder pattern like
- salt should give you diffraction at very high angle, in which case you need 
sometime to have the detector very close to the sample

That is, if those are diffracting crystals.

Agarose and/or SDS/Silver staining gels on the diluted crystals would be good 
as well. There are worth nothing as it anyway.

Cheers, leo


-
Leonard Chavas
- 
Synchrotron SOLEIL
Proxima-I
L'Orme des Merisiers
Saint-Aubin - BP 48
91192 Gif-sur-Yvette Cedex
France
- 
Phone:  +33 169 359 746
Mobile: +33 644 321 614
E-mail: leonard.cha...@synchrotron-soleil.fr
-

> On 19 Jun 2017, at 18:25, Gyanendra Kumar  wrote:
> 
> Hi Joseph,
> 
> Are you having trouble getting bigger crystals. You said you shot them, and 
> they are not salt. Do they diffract at all? If they are diffracting to any 
> extent, you could optimize the crystallization condition to get better, 
> bigger crystals and shoot them. A simple way of growing fewer, bigger 
> crystals in the same condition is to set up bigger drops and dilute the well 
> solution by water to 95%, 90%, 85%... And if that doesn't work, you can try 
> additive screen on this current crystallization condition.
> 
> As a control for determining if these are complex crystals or just DNA 
> crystals, you can set up a crystallization in the same crystallization 
> condition with just DNA, no protein.
> 
> -Gyan 
> 
> On Mon, Jun 19, 2017 at 9:20 AM, Joseph Ho  wrote:
> Dear all:
> 
> I would like to seek your opinion on our crystal hits. We are working
> on protein/dsDNA complex. By changing different protein and DNA
> (14-22bp) constructs, we recently got some hits from commercial
> screens using sitting drop vapor diffusion (very small xtals). The
> precipitant is PEG and the picture of crystals are attached. In this
> particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
> 100mM NaCl. The crystal seems floating and sit in the bottom. We do
> some test shot from other conditions and it is not salt crystals. The
> crystals can suck in izit dye.  I do some google and it seems izit dye
> also turns dsDNA crystal into blue. We also do UV/Vis microscope but
> no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.
> 
> This is our first time to work on protein/DNA complex crystals and we
> are not certain if this is just DNA or protein/DNA crystals. Can you
> provide your comments on our hits?
> 
> Thank you for your help
> 
> Joseph
> 
> 
> 
> -- 
> Gyanendra Kumar, PhD
> Associate Scientist,
> St. Jude Children's Research Hospital,
> Department of Structural Biology,
> 262, Danny Thomas Place, MS-311
> Memphis, TN 38105
> Cell: 631-875-9189
> https://www.linkedin.com/in/gyanendrakumar
> https://scholar.google.com/gyanendrakumar
> https://www.researchgate.net/profile/Gyanendra_Kumar3
> http://stjude.academia.edu/GyanendraKumar
> ---

Re: [ccp4bb] Protein or DNA crystals

[Gyanendra Kumar]

Hi Joseph,

Are you having trouble getting bigger crystals. You said you shot them, and
they are not salt. Do they diffract at all? If they are diffracting to any
extent, you could optimize the crystallization condition to get better,
bigger crystals and shoot them. A simple way of growing fewer, bigger
crystals in the same condition is to set up bigger drops and dilute the
well solution by water to 95%, 90%, 85%... And if that doesn't work, you
can try additive screen on this current crystallization condition.

As a control for determining if these are complex crystals or just DNA
crystals, you can set up a crystallization in the same crystallization
condition with just DNA, no protein.

-Gyan

On Mon, Jun 19, 2017 at 9:20 AM, Joseph Ho  wrote:

> Dear all:
>
> I would like to seek your opinion on our crystal hits. We are working
> on protein/dsDNA complex. By changing different protein and DNA
> (14-22bp) constructs, we recently got some hits from commercial
> screens using sitting drop vapor diffusion (very small xtals). The
> precipitant is PEG and the picture of crystals are attached. In this
> particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
> 100mM NaCl. The crystal seems floating and sit in the bottom. We do
> some test shot from other conditions and it is not salt crystals. The
> crystals can suck in izit dye.  I do some google and it seems izit dye
> also turns dsDNA crystal into blue. We also do UV/Vis microscope but
> no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.
>
> This is our first time to work on protein/DNA complex crystals and we
> are not certain if this is just DNA or protein/DNA crystals. Can you
> provide your comments on our hits?
>
> Thank you for your help
>
> Joseph
>



-- 
Gyanendra Kumar, PhD
Associate Scientist,
St. Jude Children's Research Hospital,
Department of Structural Biology,
262, Danny Thomas Place, MS-311
Memphis, TN 38105
Cell: 631-875-9189
https://www.linkedin.com/in/gyanendrakumar
https://scholar.google.com/gyanendrakumar

https://www.researchgate.net/profile/Gyanendra_Kumar3
http://stjude.academia.edu/GyanendraKumar
---

Re: [ccp4bb] Protein or DNA crystals

[V Nagarajan]

Hi Joseph,

Could you post the UV fluorescence image? Oftentimes, UV absorbing 
objects that do not fluoresce (such as nucleic acid crystals) show up 
dark against a brighter background in UV fluorescence.


V. Nagarajan

JANSi


On 6/19/2017 7:20 AM, Joseph Ho wrote:

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph

Re: [ccp4bb] Protein or DNA crystals

[Nicolas FOOS]

Dear Joseph,

I think, you already did the most classical test to determine what is in 
your crystal.


Maybe you can try a destructive approach by washing the crystal prior to 
put them in Agarose gel do a short run and color with ethydium bromide. 
Simpler, you can also melt the crystal after washing them and do some 
spectroscopic experiment (measurement at 260 and 280 nm) the ratio will 
help ou to know what is in your crystal.


I can add that I frequently obtained crystal of protein-DNA complex with 
this type of crystal growing condition (based on relatively small PEG).


Sometimes it was only DNA. And most of the times when it was DNA only, 
crystal have a pentagon shape, like certain one on your image.


If you have the opportunity to test diffraction, even at low resolution 
to obtain some information (cell params, matthew coeff) it will help you 
do decide what to do.


And maybe are they already good enough to do some in-situ data 
collection or serial synchrotron experiment.


Hope to help

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 19/06/2017 16:20, Joseph Ho wrote:

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph

[ccp4bb] Protein or DNA crystals

[Joseph Ho]

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph