Re: [ccp4bb] does 12 A diffraction worth optimization

[Phoebe A. Rice]

In addition to the other useful advice offered, I’d also suggest determining if 
the whole complex or just the DNA fits in the asymmetric unit.  Particularly if 
the crystals are hexagonal rods or plates, you might have DNA-only crystals.

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Natalia O 
<natalie.c...@gmail.com>
Reply-To: Natalia O <natalie.c...@gmail.com>
Date: Friday, March 2, 2018 at 7:35 PM
To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] does 12 A diffraction worth optimization

Hello,

I got crystals of protein-nucleic acid complex, rod-shape, reproducible, don’t 
visibly get damaged upon freezing; however they gave diffraction only to about 
12 A. I tried several crystals. My question is whether such crystals worth 
optimization. Clearly a 4A diffracting crystal could potentially be optimized 
to 3 – 2.5A, but if the diffraction that I am getting now is 12A it could 
suggest that the system is so flexible that getting to 3A with this crystal 
form is not possible at all. I just wonder if there is any statistics or a rule 
of thumb about what initial diffraction worth optimization?

Thank you!
-Natalia

Re: [ccp4bb] does 12 A diffraction worth optimization

[Mark J van Raaij]

it's always worth trying optimization, you never know.

also try to get a room-temperature diffraction image of your crystal, only then 
will you know if the cryo or freezing didn't damage it. If at RT it diffracts 
to high resolution, you will then know that you have to work on the 
cryo-conditions or on different mounting techniques 
(https://doi.org/10.1107/S0907444911031210 
).

RT diffraction can be done traditionally in a glass capillary, using a plastic 
capillary (like the Mitegen ones: 
https://www.mitegen.com/product/micrort-room-temperature-starter-kits/ 
), or 
in-plate, like described in this paper for instance: 
https://doi.org/10.1107/S0907444911023249 
.

Yet another option might be controlled dehydration: 
https://pubs.acs.org/doi/10.1021/cg500890r 
.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 3 Mar 2018, at 02:34, Natalia O  wrote:
> 
> Hello,
>  
> I got crystals of protein-nucleic acid complex, rod-shape, reproducible, 
> don’t visibly get damaged upon freezing; however they gave diffraction only 
> to about 12 A. I tried several crystals. My question is whether such crystals 
> worth optimization. Clearly a 4A diffracting crystal could potentially be 
> optimized to 3 – 2.5A, but if the diffraction that I am getting now is 12A it 
> could suggest that the system is so flexible that getting to 3A with this 
> crystal form is not possible at all. I just wonder if there is any statistics 
> or a rule of thumb about what initial diffraction worth optimization?
> 
> Thank you!
> -Natalia
> 

Re: [ccp4bb] does 12 A diffraction worth optimization

[Paul Miller]

Absolutely you should optimise! It's impossible to predict crystal behaviour. I 
had a 20 A crystal, and I set a new plate in the same reservoir with an 
additive screen and got a 3A crystal. It was probably a completely different 
crystal to be honest, lattice, etc, but one never knows, you just have to try. 
You can also try using the low res crystal as a seed. Plus a million other 
things.



Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Fri, 2 Mar 2018 17:34:18 -0800
>From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Natalia O 
><natalie.c...@gmail.com>)
>Subject: [ccp4bb] does 12 A diffraction worth optimization  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hello,
>
>    
>
>   I got crystals of protein-nucleic acid complex,
>   rod-shape, reproducible, don’t visibly get damaged
>   upon freezing; however they gave diffraction only to
>   about 12 A. I tried several crystals. My question is
>   whether such crystals worth optimization. Clearly a
>   4A diffracting crystal could potentially be
>   optimized to 3 – 2.5A, but if the diffraction that
>   I am getting now is 12A it could suggest that the
>   system is so flexible that getting to 3A with this
>   crystal form is not possible at all. I just wonder
>   if there is any statistics or a rule of thumb about
>   what initial diffraction worth optimization?
>
>   Thank you!
>
>   -Natalia

Re: [ccp4bb] does 12 A diffraction worth optimization

[Debanu Das]

Hi Natalia,

One rule of thumb in crystallography: anything is possible!

We have seen many cases of individual proteins (or their complexes with small 
molecules, proteins or nucleic acids) where we ended up with structures 
starting from low resolution initial crystals. So before taking a decision, I 
would suggest considering the following:

a) How many of these crystals did you screen? If only a very few, you should 
try more.
b) How big are the crystals?
c) Are these diffraction results from home source or synchrotron?
d) Long long were crystals allowed to grow before you harvested?
e) Did you try a few different cryos to verify they don't show any difference 
despite no visible deterioration?
f) How long does it take to harvest and place in cryo? How long are you soaking 
in cryo before freezing?
g) Are there issues in crystals being stuck in the well or cover slide and you 
have to apply force to dislodge?
h) Are you doing single step cryo or multi-step in increasing amounts of cryo?
i) Did you try growing crystals at different temp (4C and 20C?) or growing with 
cryo?
j) Do you know what the construct of this apo protein can diffract to?

If you have tried all the above, you can then consider trying different DNA 
lengths and blunt-end vs overhangs or different protein constructs.

Happy to discuss more about the specific case.

Best,
Debanu 
Accelero Biostructures 


> On Fri, Mar 2, 2018 at 5:34 PM, Natalia O  wrote:
> Hello,
> 
> 
> 
> I got crystals of protein-nucleic acid complex, rod-shape, reproducible,
> don’t visibly get damaged upon freezing; however they gave diffraction only
> to about 12 A. I tried several crystals. My question is whether such
> crystals worth optimization. Clearly a 4A diffracting crystal could
> potentially be optimized to 3 – 2.5A, but if the diffraction that I am
> getting now is 12A it could suggest that the system is so flexible that
> getting to 3A with this crystal form is not possible at all. I just wonder
> if there is any statistics or a rule of thumb about what initial diffraction
> worth optimization?
> 
> 
> Thank you!
> 
> -Natalia
> 
> 

[ccp4bb] does 12 A diffraction worth optimization

[Natalia O]

Hello,



I got crystals of protein-nucleic acid complex, rod-shape, reproducible,
don’t visibly get damaged upon freezing; however they gave diffraction only
to about 12 A. I tried several crystals. My question is whether such
crystals worth optimization. Clearly a 4A diffracting crystal could
potentially be optimized to 3 – 2.5A, but if the diffraction that I am
getting now is 12A it could suggest that the system is so flexible that
getting to 3A with this crystal form is not possible at all. I just wonder
if there is any statistics or a rule of thumb about what initial
diffraction worth optimization?


Thank you!

-Natalia