Re: [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone crystal?

[Philippe BENAS]

Dear Joseph,
I fully agree with Daniel Himmel's answer but you might be able to "index" your 
reflections and get a approximate cell parameters.
I did that in the past with crystals that diffracted very poorly up to 15 
angst. I used HKL200 at that time, cheated with the distance (HKL2000 won't 
accept indexing with too low resolution spots) and selected individual spots 
manually.It was enough for making Matthews analysis for each putative space 
group.The complex formation between the protein and the RNA was confirmed by a 
stoechiometric amount of each macromolecule resulting from OD spectra 
deconvolution taken on dissolved crystals, using a spectrum for the protein 
alone and a spectrum for the RNA alone as references.
Best regards,
Philippe Philippe BENAS, Ph.D.

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18



  De : Joseph Ho <sbddintai...@gmail.com>
 À : CCP4BB@JISCMAIL.AC.UK 
 Envoyé le : Lundi 12 mars 2018 12h54
 Objet : [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone 
crystal?
   
Dear all:


I would like to seek your wisdom on our latest diffraction pattern. We
have been working on protein/DNA complex. The protein and DNA have
similar MW. By binding assay, we know the minimal length of DNA. (The
Kd is 0.1-1 microMolar and we can see the complex formation in size
exclusion chromatography up to 200mM NaCl but also some unbound form)
After trying different length of DNA, we recently obtained many
crystal hits (the percipient is either PEG400 or MPD). The final ratio
(prior to protein crystallization) between protein and DNA is 1:1.6
considering some loss of protein during concentration. The crystal is
birefringent. Since high conc. of PEG400 (MPD), the crystals were
directly frozen in liquid N2. However, crystals only diffract to 8-10
angstrom (anisotropic) and also  weird striking line are present
(please see attachment). Do you think if it is  DNA alone crystal or
protein/DNA complex crystal?
How should I improve the diffraction quality?



PS. We have done some tests. For example, set up the same conditions
with DNA alone. I also tried to dissolve crystals in Bradford assay
solution and I believe I saw some blueish color. But none of these
tests are conclusive.

Thanks for your suggestion.

Joseph


   

Re: [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone crystal?

[Daniel M. Himmel, Ph. D.]

The diffraction patterns clearly show an overlap of two or more

lattices, which either means you didn’t have a single crystal at the

start or it was damaged during the flash-cooling process.  PEG 400

is generally a good cryoprotectant at ≥20%, but I would recommend using

at least 25% to be certain no ice formation damages the crystal lattice.


The high mosaicity makes interpretation of the diffraction pattern open to

debate.  Some of the reflections look like they could originate from
protein,

since they indicate long distance repeats.  Some double-spots suggest a
cracked

crystal with high mosaicity.  It’s a difficult call for me, but I think the
layer

lines look roughly like an hour glass shape, which is characteristically

seen in DNA patterns and arises from the double-helical structure.

Again, the layer lines suggest at least 2 or 3 lattices.  To pin down

whether you’re really looking at double-helical DNA, you need to

measure the real space distance suggested from layer lines (measure

the equiv. resolution from the reciprocal space origin).  I don’t have

time to look it up now, but there should be tell-tale layer line repeats

(if I recall correctly) at about 10 A and 34 A.  You can look for some

old papers by Francis Crick on helical theory (sorry I don’t have

the reference at my fingertips).  In summary, I would cautiously

surmise that you have a protein (small round spot reflections), bound

by DNA that is much more mobile (highly mosaic layer lines), and that

both the protein and DNA exist in multiple (probably three) lattices, but,

again, the distance repeats of the layer lines have to be measured to

see if they are consistent with double-helical DNA.


Try to get crystals that diffract better for more clarity.  Flash cool in

25% PEG 400 (or try combination of PEG 400 and PEG 200), or

vary the MPD concentration.  Good luck.


I hope this helps.


-Daniel



[image:
RnNZfQn2o2xpggJQqefCOervMbPIci5mujDPJnvl43kv6Rtxjyh5gHN_JKVzeU-aaGz3pePFgxfoAAtZJZNx8mveVTc-11j98EfuAJVcumUenA=s0-d-e1-ft.gif]Daniel
M. Himmel, Ph. D.

URL:  http://www.DanielMHimmel.com 


On Mon, Mar 12, 2018 at 11:24 AM, Joseph Ho  wrote:

> Dear Xiao and Hans:
>
> Thanks for your reply. We tried to index it but failed.
>
> Joseph
> On Mon, Mar 12, 2018 at 11:15 PM, Xiao Lei  wrote:
> > did you try to index it?  the cell dimensions may give you hint.
> >
> > On Mon, Mar 12, 2018 at 4:40 AM, Joseph Ho 
> wrote:
> >>
> >> Dear all:
> >>
> >>
> >> I would like to seek your wisdom on our latest diffraction pattern. We
> >> have been working on protein/DNA complex. The protein and DNA have
> >> similar MW. By binding assay, we know the minimal length of DNA. (The
> >> Kd is 0.1-1 microMolar and we can see the complex formation in size
> >> exclusion chromatography up to 200mM NaCl but also some unbound form)
> >> After trying different length of DNA, we recently obtained many
> >> crystal hits (the percipient is either PEG400 or MPD). The final ratio
> >> (prior to protein crystallization) between protein and DNA is 1:1.6
> >> considering some loss of protein during concentration. The crystal is
> >> birefringent. Since high conc. of PEG400 (MPD), the crystals were
> >> directly frozen in liquid N2. However, crystals only diffract to 8-10
> >> angstrom (anisotropic) and also  weird striking line are present
> >> (please see attachment). Do you think if it is  DNA alone crystal or
> >> protein/DNA complex crystal?
> >> How should I improve the diffraction quality?
> >>
> >>
> >>
> >> PS. We have done some tests. For example, set up the same conditions
> >> with DNA alone. I also tried to dissolve crystals in Bradford assay
> >> solution and I believe I saw some blueish color. But none of these
> >> tests are conclusive.
> >>
> >> Thanks for your suggestion.
> >>
> >> Joseph
> >
> >
>

Re: [ccp4bb] Strange Diffraction pattern! Protein/DNA complex or DNA alone crystal?

[Joseph Ho]

Dear Xiao and Hans:

Thanks for your reply. We tried to index it but failed.

Joseph
On Mon, Mar 12, 2018 at 11:15 PM, Xiao Lei  wrote:
> did you try to index it?  the cell dimensions may give you hint.
>
> On Mon, Mar 12, 2018 at 4:40 AM, Joseph Ho  wrote:
>>
>> Dear all:
>>
>>
>> I would like to seek your wisdom on our latest diffraction pattern. We
>> have been working on protein/DNA complex. The protein and DNA have
>> similar MW. By binding assay, we know the minimal length of DNA. (The
>> Kd is 0.1-1 microMolar and we can see the complex formation in size
>> exclusion chromatography up to 200mM NaCl but also some unbound form)
>> After trying different length of DNA, we recently obtained many
>> crystal hits (the percipient is either PEG400 or MPD). The final ratio
>> (prior to protein crystallization) between protein and DNA is 1:1.6
>> considering some loss of protein during concentration. The crystal is
>> birefringent. Since high conc. of PEG400 (MPD), the crystals were
>> directly frozen in liquid N2. However, crystals only diffract to 8-10
>> angstrom (anisotropic) and also  weird striking line are present
>> (please see attachment). Do you think if it is  DNA alone crystal or
>> protein/DNA complex crystal?
>> How should I improve the diffraction quality?
>>
>>
>>
>> PS. We have done some tests. For example, set up the same conditions
>> with DNA alone. I also tried to dissolve crystals in Bradford assay
>> solution and I believe I saw some blueish color. But none of these
>> tests are conclusive.
>>
>> Thanks for your suggestion.
>>
>> Joseph
>
>