Re: [ccp4bb] Crystals with DNA

[Guillaume Gaullier]

Hello Careina,

In addition to all the good suggestions you already got, I can point to these 
very good references:

- Pryor EE Jr, Wozniak DJ & Hollis T (2012) Crystallization of Pseudomonas 
aeruginosa AmrZ protein: development of a comprehensive method for obtaining 
and optimization of protein-DNA crystals. Acta Crystallogr Sect F Struct Biol 
Cryst Commun 68: 985–993 https://doi.org/10.1107/S1744309112025316

- Hollis T (2007) Crystallization of protein-DNA complexes. Methods Mol Biol 
363: 225–237 https://doi.org/10.1007/978-1-59745-209-0_11

- And these lecture notes also have very useful info: 
https://gradebuddy.com/doc/2627393/protein-dna-complexes/
(Now I can’t find the link on the author’s website, where I originally found 
it…)

The 2012 paper presents a PEG-salt screen of 48 conditions rationally designed 
for protein-DNA complexes. It is commercially available (I can’t find the 
company selling it now), but also not difficult to prepare from scratch. Might 
be worth a try if the conditions in this screen differ significantly from your 
current best conditions.

The 2007 review has very good advice on all the critical points specific to 
protein-DNA complexes, and suggestions for DNA construct design. If you are on 
a budget, and in any case if you want overhangs, you should buy single-stranded 
oligonucleotides and anneal them yourself. One way to minimize the number of 
oligos you need to buy is to design one top strand with the sequence you want, 
and three different bottom strands: on that will make blunt ends, one that will 
make 5’-overhangs and one that will make 3’-overhangs. Ideally you want to test 
the effect of overhangs, so you don’t want to change the base composition. The 
easy way is to remove one letter from the 3’-end and put it back at the other 
end (for the 5’-overhang) and vice versa (for the 3’-overhang).
I don’t remember where I read this (likely in one of these references), but it 
seems that sticky ends with a 1-bp overhang and A/T pairing are better at 
promoting crystallization than sticky ends with longer overhangs and ending on 
a G/C pairing (somewhat counter intuitively).

Another thing to optimize is the length of the DNA. You might be constrained by 
other factors, mainly the length of the motif the protein binds to. But also 
consider that, when looking at all crystal structures of protein-DNA complexes 
in the PDB, it is apparent that certain DNA lengths are much more common, with 
12 and 16 bp largely over-represented, presumably because different lengths 
crystallize less easily (or because people solving such structures don’t 
deposit them…). See the histogram of number of PDB entries by DNA length here: 
https://guillawme.github.io/insights-from-the-pdb/dna-length-in-protein-dna-complexes.html#0-150_bp_range
This bias in DNA length is even more pronounced in crystals of free DNA, with 
6, 8, 10 and 12 bp vastly over-represented: 
https://guillawme.github.io/insights-from-the-pdb/free-dna.html#DNA_length_in_crystal_structures_of_free_DNA
So, for free DNA, an even number of base pairs and somewhere around an integer 
number of DNA turns (~10 bp) seem most favored for crystallization. Once you 
add a protein, you have to take into account the length of the binding motif, 
and if you decide to design an oligo with two binding motifs then you also need 
to think about the spacing between the two (if it’s not constrained by the 
binding mode of the protein) because this spacing will also of course affect 
the relative rotation between the two bound proteins around the DNA axis, which 
might affect packing.

(I last updated these histograms in December, so this is relatively fresh data.)

I hope this helps,

Guillaume


On 9 Feb 2024, at 13:09, Patrick Shaw Stewart 
mailto:patr...@douglas.co.uk>> wrote:

Carina, to complement the techniques using sticky ends, etc., you can also use 
the "random" microseeding approach that I mentioned to Kavya, see below.

The great advantage in a project like yours, where you have a family of related 
constructs, is that you can use cross-seeding - that is, you can use crushed 
crystals of one construct to seed other target constructs.  You can even mix 
several seed stocks together, although we always keep seed crystals grown in 
high-salt conditions separate from those grown in high-peg conditions.

There are some very nice examples of cross-seeding and mixing seed stocks in 
this paper by Obmolova et al.

Obmolova, G., Malia, T.J., Teplyakov, A., Sweet, R.W. and Gilliland, G.L., 
2014. Protein crystallization with microseed matrix screening: application to 
human germline antibody Fabs. Acta Crystallographica Section F: Structural 
Biology Communications, 70(8), pp.1107-1115.  
https://doi.org/10.1107/S2053230X14012552

More info

https://www.douglas.co.uk/mms.htm

Best wishes and good luck!

Patrick
___

Hi Kavya

1. Make a seed stock from the globules or anything else that you think might be 

Re: [ccp4bb] Crystals with DNA

[Patrick Shaw Stewart]

Carina, to complement the techniques using sticky ends, etc., you can also
use the "random" microseeding approach that I mentioned to Kavya, see below.

The great advantage in a project like yours, where you have a family of
related constructs, is that you can use cross-seeding - that is, you can
use crushed crystals of one construct to seed other target constructs.  You
can even mix several seed stocks together, although we always keep seed
crystals grown in high-salt conditions separate from those grown in
high-peg conditions.

There are some very nice examples of cross-seeding and mixing seed stocks
in this paper by Obmolova et al.

Obmolova, G., Malia, T.J., Teplyakov, A., Sweet, R.W. and Gilliland, G.L.,
2014. Protein crystallization with microseed matrix screening: application
to human germline antibody Fabs. *Acta Crystallographica Section F:
Structural Biology Communications*, *70*(8), pp.1107-1115.
https://doi.org/10.1107/S2053230X14012552


More info

https://www.douglas.co.uk/mms.htm


Best wishes and good luck!

Patrick
___

Hi Kavya

1. Make a seed stock from the globules or anything else that you think
might be crystalline, and recreen.  In other words, you should add your
seed stock to *random screens* (not optimization experiments).  There could
be many conditions that are in the metastable zone of the phase diagram in
your normal screens - this method can give you crystals in those conditions.

If this works, you'll be in a better position anyway because you'll have
more control - by diluting the seed stock, you can control the number of
crystals per drop.

References:


D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed
matrix-screening method for protein crystallization. *Acta
Crystallographica Section D: Biological Crystallography*, *63*(4),
pp.550-554.

Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock,
P.F., 2011. Random microseeding: a theoretical and practical exploration of
seed stability and seeding techniques for successful protein
crystallization. *Crystal Growth & Design*, *11*(8), pp.3432-3441.


This is how we normally make the seed:


https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf




On Thu, Feb 8, 2024 at 11:26 AM careinaedgo...@yahoo.com <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

>  Hello all.
>
> I am struggling to get defracting crystals with a protein DNA complex. The
> crystals are plentiful but they do not diffract. I am going back to the
> grind stone and relookong at my DNA sequence.
> Is there any wisdom you could give me with regards to what works best with
> DNA in crystals?
> From my reading it seems if the length is a multiple of 7 (for B DNA) and
> blunt ended, it will stretch over the length of the crystal and improve
> crystalisability. But if you want crystals that diffract better, you will
> need to play with length and even making it only one base longer or shorter
> can make a difference, even changing the morphology of the crystal? Longer
> is better than shorter, and overhangs are good for improving diffraction?
> Presumably because they stabilize contacts? It is expensive to synthesize a
> while bunch of sequences so I need to be strategic in my choice. Would
> appreciate any advice.
> Thank you
> Careina.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
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 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek

 http://www.douglas.co.uk
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Re: [ccp4bb] Crystals with DNA

[Fred Vellieux]

Hello,

Overhanging "sticky" ends are mentioned frequently when it comes to 
obtaining infinite helices that are useful in crystallization. For 
example in 
https://home.ccr.cancer.gov/csb/nihxray/Tips-and-Tricks_Crystallization_Protein-DNA_updated.pdf 
.


Cheers,

Fred.

On 09/02/2024 10:59, careinaedgo...@yahoo.com wrote:

Thank you for this insight, Nicolas. It is very helpful.
Yes I have also had a soccer ball shaped crystal that does not 
diffract as well as, and more recently, many plate like crystals but 
they do not diffract either.
I do know I have both protein and DNA in my crystals but I do not 
know, as you say, exactly what is forming the crystal contacts.
Just to be clear, do you say overhangs are helpful? Surely overhangs 
won't promote an infinite helix? If one wants an infinite helix, would 
the DNA not have to be blunt ended?


Sent from Yahoo Mail on Android 



On Thu, Feb 8, 2024 at 4:59 PM, Nicolas Foos
 wrote:

Hello Careina,

In my hands, DNA protein complex crystals may be frustrating, 
because often we get good looking crystals which don't diffract at
all and are actually not easy to improve.

I remember obtaining a lot of crystal looking a bit like "STOP"
road sign (octogonal shape for one axis) which never diffracts.
(Often containing only DNA not well organized)

So long story short, In my hand (transciption factor bound with
homeodomain for example). I had good results with DNA sequence
which results in hoverhangs. The idea was to bet on a "infinit"
DNA helix which should help the packing.

I strongly encouraged you to rely on any other information  you
can have to be sure of what is the best minimal sequence (like
band shift assay). Also if you can purify the entire complex
before crystallization assay (I don't know your protocol, but
ideally, I would prepare the complex prot-DNA and put it on size
exclusion).

The point is, you don't know /a priori /what kind of crystal
packing you will have. It may be only due to protein protein
contact and not related to the DNA directly.

Also, I often get good results with crystal growing condition
containing MPD or PEG (makes me using PEG screen Familly as first
approach).

I invite you to read the Timothy Richmond teams Papers on 
nucleosome they spend some times improving the resolution on very
large complex. (Luger etal 1997).

There is many parameters, DNA sequence also change a bit the DNA
geometry (look for A-tract), You may want to introduce such
sequence to maybe improve the "rigidity".

Also if your DNA fragment are small, be careful with the
temperature. The annealing and the DNA duplex formation is
critical and you should be careful on your procedure.

I remember that small cation like Li, may help too.

HTH


Nicolas


On 08/02/2024 12:25, careinaedgo...@yahoo.com
 wrote:
 Hello all.

I am struggling to get defracting crystals with a protein DNA
complex. The crystals are plentiful but they do not diffract. I am
going back to the grind stone and relookong at my DNA sequence.
Is there any wisdom you could give me with regards to what works
best with DNA in crystals?
From my reading it seems if the length is a multiple of 7 (for B
DNA) and blunt ended, it will stretch over the length of the
crystal and improve crystalisability. But if you want crystals
that diffract better, you will need to play with length and even
making it only one base longer or shorter can make a difference,
even changing the morphology of the crystal? Longer is better than
shorter, and overhangs are good for improving diffraction?
Presumably because they stabilize contacts? It is expensive to
synthesize a while bunch of sequences so I need to be strategic in
my choice. Would appreciate any advice.
Thank you
Careina.



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-- 
Nicolas Foos PhD - ARISE fellow

https://orcid.org/-0003-2331-8399  


EMBL Grenoble, McCarthy Team

71 av. des Martyrs,
38000 Grenoble FRANCE

+33 4 57 42 84 67





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Re: [ccp4bb] Crystals with DNA

[Nicolas Foos]

Hello,

If you want to promote "infinite" helix, you should go for overhangs 
(sticky end)  with compatible sequence. I try to explain better what I 
have in mind (and I actually did).


example :

5'ATCCCTAAATCGGCGTGTGCT---3'

3'---GGATTTAGCCGCACACGATAG5'

Hoping that this results in something like :

5' ATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCT---3'

3' ---GGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAG5'

With the blunt end, you may have the helix, or not, in my opinion your 
are not really promoting the "infinite helix".


Nicolas

On 09/02/2024 10:59, careinaedgo...@yahoo.com wrote:

Thank you for this insight, Nicolas. It is very helpful.
Yes I have also had a soccer ball shaped crystal that does not 
diffract as well as, and more recently, many plate like crystals but 
they do not diffract either.
I do know I have both protein and DNA in my crystals but I do not 
know, as you say, exactly what is forming the crystal contacts.
Just to be clear, do you say overhangs are helpful? Surely overhangs 
won't promote an infinite helix? If one wants an infinite helix, would 
the DNA not have to be blunt ended?


Sent from Yahoo Mail on Android 



On Thu, Feb 8, 2024 at 4:59 PM, Nicolas Foos
 wrote:

Hello Careina,

In my hands, DNA protein complex crystals may be frustrating, 
because often we get good looking crystals which don't diffract at
all and are actually not easy to improve.

I remember obtaining a lot of crystal looking a bit like "STOP"
road sign (octogonal shape for one axis) which never diffracts.
(Often containing only DNA not well organized)

So long story short, In my hand (transciption factor bound with
homeodomain for example). I had good results with DNA sequence
which results in hoverhangs. The idea was to bet on a "infinit"
DNA helix which should help the packing.

I strongly encouraged you to rely on any other information  you
can have to be sure of what is the best minimal sequence (like
band shift assay). Also if you can purify the entire complex
before crystallization assay (I don't know your protocol, but
ideally, I would prepare the complex prot-DNA and put it on size
exclusion).

The point is, you don't know /a priori /what kind of crystal
packing you will have. It may be only due to protein protein
contact and not related to the DNA directly.

Also, I often get good results with crystal growing condition
containing MPD or PEG (makes me using PEG screen Familly as first
approach).

I invite you to read the Timothy Richmond teams Papers on 
nucleosome they spend some times improving the resolution on very
large complex. (Luger etal 1997).

There is many parameters, DNA sequence also change a bit the DNA
geometry (look for A-tract), You may want to introduce such
sequence to maybe improve the "rigidity".

Also if your DNA fragment are small, be careful with the
temperature. The annealing and the DNA duplex formation is
critical and you should be careful on your procedure.

I remember that small cation like Li, may help too.

HTH


Nicolas


On 08/02/2024 12:25, careinaedgo...@yahoo.com
 wrote:
 Hello all.

I am struggling to get defracting crystals with a protein DNA
complex. The crystals are plentiful but they do not diffract. I am
going back to the grind stone and relookong at my DNA sequence.
Is there any wisdom you could give me with regards to what works
best with DNA in crystals?
From my reading it seems if the length is a multiple of 7 (for B
DNA) and blunt ended, it will stretch over the length of the
crystal and improve crystalisability. But if you want crystals
that diffract better, you will need to play with length and even
making it only one base longer or shorter can make a difference,
even changing the morphology of the crystal? Longer is better than
shorter, and overhangs are good for improving diffraction?
Presumably because they stabilize contacts? It is expensive to
synthesize a while bunch of sequences so I need to be strategic in
my choice. Would appreciate any advice.
Thank you
Careina.



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


-- 
Nicolas Foos PhD - ARISE fellow

https://orcid.org/-0003-2331-8399  


EMBL Grenoble, McCarthy Team

71 av. des Martyrs,
38000 Grenoble 

Re: [ccp4bb] Crystals with DNA

[careinaedgo...@yahoo.com]

Thank you for this insight, Nicolas. It is very helpful.Yes I have also had a 
soccer ball shaped crystal that does not diffract as well as, and more 
recently, many plate like crystals but they do not diffract either.I do know I 
have both protein and DNA in my crystals but I do not know, as you say, exactly 
what is forming the crystal contacts.Just to be clear, do you say overhangs are 
helpful? Surely overhangs won't promote an infinite helix? If one wants an 
infinite helix, would the DNA not have to be blunt ended?

Sent from Yahoo Mail on Android 
 
  On Thu, Feb 8, 2024 at 4:59 PM, Nicolas Foos wrote:
Hello Careina, 
 
 
In my hands, DNA protein complex crystals may be frustrating,  because often we 
get good looking crystals which don't diffract at all and are actually not easy 
to improve. 
 
 
I remember obtaining a lot of crystal looking a bit like "STOP" road sign 
(octogonal shape for one axis) which never diffracts. (Often containing only 
DNA not well organized)
 
 
So long story short, In my hand (transciption factor bound with homeodomain for 
example). I had good results with DNA sequence which results in hoverhangs. The 
idea was to bet on a "infinit" DNA helix which should help the packing. 
 
 
I strongly encouraged you to rely on any other information  you can have to be 
sure of what is the best minimal sequence (like band shift assay). Also if you 
can purify the entire complex before crystallization assay (I don't know your 
protocol, but ideally, I would prepare the complex prot-DNA and put it on size 
exclusion).
 
The point is, you don't know a priori what kind of crystal packing you will 
have. It may be only due to protein protein contact and not related to the DNA 
directly. 
 
Also, I often get good results with crystal growing condition containing MPD or 
PEG (makes me using PEG screen Familly as first approach). 
 
 
I invite you to read the Timothy Richmond teams Papers on  nucleosome they 
spend some times improving the resolution on very large complex. (Luger etal 
1997).
 
There is many parameters, DNA sequence also change a bit the DNA geometry (look 
for A-tract), You may want to introduce such sequence to maybe improve the 
"rigidity". 
 
 
Also if your DNA fragment are small, be careful with the temperature. The 
annealing and the DNA duplex formation is critical and you should be careful on 
your procedure.
 
 
I remember that small cation like Li, may help too.  
 
 
HTH 
 
 

 
 
Nicolas
 
 

 
 On 08/02/2024 12:25, careinaedgo...@yahoo.com wrote:
  
 
  Hello all. 
  I am struggling to get defracting crystals with a protein DNA complex. The 
crystals are plentiful but they do not diffract. I am going back to the grind 
stone and relookong at my DNA sequence. Is there any wisdom you could give me 
with regards to what works best with DNA in crystals? From my reading it seems 
if the length is a multiple of 7 (for B DNA) and blunt ended, it will stretch 
over the length of the crystal and improve crystalisability. But if you want 
crystals that diffract better, you will need to play with length and even 
making it only one base longer or shorter can make a difference, even changing 
the morphology of the crystal? Longer is better than shorter, and overhangs are 
good for improving diffraction? Presumably because they stabilize contacts? It 
is expensive to synthesize a while bunch of sequences so I need to be strategic 
in my choice. Would appreciate any advice. Thank you Careina.  
  
To unsubscribe from the CCP4BB list, click the following link:
 https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
 -- 
Nicolas Foos PhD - ARISE fellow
https://orcid.org/-0003-2331-8399
   
EMBL Grenoble, McCarthy Team
71 av. des Martyrs, 
38000 Grenoble FRANCE
   
+33 4 57 42 84 67 

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Re: [ccp4bb] Crystals with DNA

[Nicolas Foos]

Hello Careina,

In my hands, DNA protein complex crystals may be frustrating, because 
often we get good looking crystals which don't diffract at all and are 
actually not easy to improve.


I remember obtaining a lot of crystal looking a bit like "STOP" road 
sign (octogonal shape for one axis) which never diffracts. (Often 
containing only DNA not well organized)


So long story short, In my hand (transciption factor bound with 
homeodomain for example). I had good results with DNA sequence which 
results in hoverhangs. The idea was to bet on a "infinit" DNA helix 
which should help the packing.


I strongly encouraged you to rely on any other information  you can have 
to be sure of what is the best minimal sequence (like band shift 
assay). Also if you can purify the entire complex before crystallization 
assay (I don't know your protocol, but ideally, I would prepare the 
complex prot-DNA and put it on size exclusion).


The point is, you don't know /a priori /what kind of crystal packing you 
will have. It may be only due to protein protein contact and not related 
to the DNA directly.


Also, I often get good results with crystal growing condition containing 
MPD or PEG (makes me using PEG screen Familly as first approach).


I invite you to read the Timothy Richmond teams Papers on nucleosome 
they spend some times improving the resolution on very large complex. 
(Luger etal 1997).


There is many parameters, DNA sequence also change a bit the DNA 
geometry (look for A-tract), You may want to introduce such sequence to 
maybe improve the "rigidity".


Also if your DNA fragment are small, be careful with the temperature. 
The annealing and the DNA duplex formation is critical and you should be 
careful on your procedure.


I remember that small cation like Li, may help too.

HTH


Nicolas


On 08/02/2024 12:25, careinaedgo...@yahoo.com wrote:

 Hello all.

I am struggling to get defracting crystals with a protein DNA complex. 
The crystals are plentiful but they do not diffract. I am going back 
to the grind stone and relookong at my DNA sequence.
Is there any wisdom you could give me with regards to what works best 
with DNA in crystals?
From my reading it seems if the length is a multiple of 7 (for B DNA) 
and blunt ended, it will stretch over the length of the crystal and 
improve crystalisability. But if you want crystals that diffract 
better, you will need to play with length and even making it only one 
base longer or shorter can make a difference, even changing the 
morphology of the crystal? Longer is better than shorter, and 
overhangs are good for improving diffraction? Presumably because they 
stabilize contacts? It is expensive to synthesize a while bunch of 
sequences so I need to be strategic in my choice. Would appreciate any 
advice.

Thank you
Careina.



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




--
Nicolas Foos PhD - ARISE fellow
https://orcid.org/-0003-2331-8399
   
EMBL Grenoble, McCarthy Team

71 av. des Martyrs,
38000 Grenoble FRANCE
   
+33 4 57 42 84 67




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[ccp4bb] RES: [ccp4bb] Crystals with DNA

[Rafael Marques]

Hi Careina.

The top two things that come to my mind is that you need to screen for 
different crystallization conditions and do some seeding. If your Crystal 
conditions does not diffract at all, probably it won’t.  Have you tried to 
optimize your condintion changing the pH or precipitant concentration?

Best wishes


Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester

Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"



De: CCP4 bulletin board  em nome de 
careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
Enviado: Thursday, February 8, 2024 8:25:25 AM
Para: CCP4BB@JISCMAIL.AC.UK 
Assunto: [ccp4bb] Crystals with DNA

 Hello all.

I am struggling to get defracting crystals with a protein DNA complex. The 
crystals are plentiful but they do not diffract. I am going back to the grind 
stone and relookong at my DNA sequence.
Is there any wisdom you could give me with regards to what works best with DNA 
in crystals?
>From my reading it seems if the length is a multiple of 7 (for B DNA) and 
>blunt ended, it will stretch over the length of the crystal and improve 
>crystalisability. But if you want crystals that diffract better, you will need 
>to play with length and even making it only one base longer or shorter can 
>make a difference, even changing the morphology of the crystal? Longer is 
>better than shorter, and overhangs are good for improving diffraction? 
>Presumably because they stabilize contacts? It is expensive to synthesize a 
>while bunch of sequences so I need to be strategic in my choice. Would 
>appreciate any advice.
Thank you
Careina.



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



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[ccp4bb] Crystals with DNA

[careinaedgo...@yahoo.com]

 Hello all.
I am struggling to get defracting crystals with a protein DNA complex. The 
crystals are plentiful but they do not diffract. I am going back to the grind 
stone and relookong at my DNA sequence.Is there any wisdom you could give me 
with regards to what works best with DNA in crystals?From my reading it seems 
if the length is a multiple of 7 (for B DNA) and blunt ended, it will stretch 
over the length of the crystal and improve crystalisability. But if you want 
crystals that diffract better, you will need to play with length and even 
making it only one base longer or shorter can make a difference, even changing 
the morphology of the crystal? Longer is better than shorter, and overhangs are 
good for improving diffraction? Presumably because they stabilize contacts? It 
is expensive to synthesize a while bunch of sequences so I need to be strategic 
in my choice. Would appreciate any advice.Thank youCareina.



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