Thank you so much guys for all the suggestions. I will definitely try some of
them. Just telling you a bit more about my case, especially the average size
crystal:
- I screened for different protein concentration (15, 10 and 5 mg/mL),
different precipitant/protein ratio (2:1, 1:1 and 1:2) and also different room
temperatures (4, 10 and 20ºC). I also tried different commercial kits
(Morpheus, BCS, SG1, PACT and INDEX)
- I tried co crystallization with some of its bindings too (GTP, GDP and
GTP-y)...no improvement
- As cryo protector I used glycerol, PEG200 and PEG400 (no difference
concerning diffraction)
- I screened manually a range of pH and PEG percentage from my best condition
(the biggest crystals), even changed the volume of the drops and the reservoir
solution (sometimes I didn’t even add it)
- the crystals are rod-like...pointless tells me they are P63 2 2 and I
couldn’t get another space group till now. And yes, there is a lot of solvent
(~79%) and I believe that this is behind with this protein crystal don’t
diffract beyond the necessary.
Thank you very much again, guys
Best regards
Rafael Marques da Silva
Mestrando 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: Andrew Purkiss<mailto:a.purk...@mail.cryst.bbk.ac.uk>
Enviado:sexta-feira, 18 de dezembro de 2020 12:50
Para: Rafael Marques<mailto:rafael_mmsi...@hotmail.com>;
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: Re: [ccp4bb] Micro/Macro crystal seeding experience
We recently had a projects where removing the HIS-tag changed an
unsolvable crystal form into one where Molecular Replacement worked
immediately (and with an NMR ensemble as the search model to boot). In
this case the HIS-tag was about 5% of the total domain mass and the
tags were probably tangled up in the centre of the unit cell.
Along with all the other suggestions, I would also put a word in for
crystallisation at a lower/different temperature. We've had several
projects where this has improved diffraction from worse than 4 Ang. to
better than 2 Ang. or where different forms have grown. You can try all
the other suggestions in the cold room as well as well as your normal
condition.
I've been trying to get all our users to screen new crystal forms (both
new projects and new conditions in existing projects) in-situ, before
trying cryo-cooled data collection. This gives a baseline before
opening the drops for cryo-protection and cryo-cooling and may also
give some guidelines for dehydration etc.
Good luck.
Andy Purkiss
On Thu, 2020-12-17 at 21:30 +0000, Rafael Marques wrote:
> Hello all, how are you doing?
>
> I have been working with two different proteins for 3 years. I was
> able to purify them by IMAC and SEC, no problems at all, and I have
> got some crystals of both. However, the best data I have got until
> now is around 4A. The diffraction experiment was carried out several
> times at DLS.
>
> The crystals for each protein are different. One is really small
> whereas the other one is a regular size (Images below). Some people
> have told me to try microseeding and I did this, but with no success
> at all.
>
> So I am asking here if there is someone experienced in this kind of
> witchcraft that could give me a hand and also answer me these two
> things:
>
> Have you ever had good size crystals that didn’t diffract beyond 4A
> and after trying seeding you have got very good diffraction data (in
> these case with no change concerning the crystal size)?
>
> What is the best protocol that you have tried to increase the size of
> your crystals and make them diffract better?
>
> Many thanks in advance.
>
> Best
>
>
>
>
> Rafael Marques da Silva
> Mestrando 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"
> ________________________________________________
>
>
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--
Andrew Purkiss
Structural Biology STP
The Francis Crick Institute
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