Hi there,

The answer is there is actually no way to know. Cristallogenesis is a purely empiric technique. The only thing you have to be sure of is the homogeneity of your sample and to make sure it is concentrated enough. If you get absolutely no hit, I think you have to improve/change the protocol for the preparation of the sample. If you get precipitates it shows at least that the concentration of the sample is appropriate.

Hi Rakesh,

crystallization is a trail-and-error process I’m afraid, but there are still some

things you could try, e.g:

-optimize buffer composition (where your protein is stable) using ThermoFluor or ThermoFAD assay-find more details here: http://www.beta-sheet.org/resources/T20-EMBL-Hamburg.pdf

-do a Pre-Crystallization test, e.g. from Hampton Research (https://www.hamptonresearch.com/product_detail.aspx?cid=1&sid=29&pid=10)

-use online servers to inspire your mutagenesis efforts, e.g: SERp Server (http://services.mbi.ucla.edu/SER/)

or XtalPred (http://ffas.burnham.org/XtalPred-cgi/xtal.pl)

-use DLS (Dynamic Light Scattering) to determine the aggregation state of your protein

-perhaps use of different expression system (e.g. different tag, different position of a tag)

HTH, Ilona

Hi

I agree with Dominique Liger answers

good luck

Thank you everyone for your kind suggestions. I will try them and hope to get crystals.

Sorry for few more questions. The protein I am working is about 55 kD and well expressed in E. coli, soluble and could be purified with common process of chromatographic techniques. Should I add a tag protein? Does a tag protein help to increase the probability of crystallization?

it is not highly convincing an reliable but what about CD analysis of your protein ?

The tag usually does not help the crystallization. The opposite is often the case that the tag hinders crystallization. However that is only my own experience.

I would recommand the following:

a) as suggested optimize the buffer conditions

b) make sure that your protein is pure (95% or higher is recommanded)

c) check is your protein is monodispers (this increases chances of crystallization in my experience)

d) increase protein concentration for crystallization (I optimize for roughly 80-90% precipitation in the initial screen) - if you see still clear drops your concentration might be too low!

e) try limited proteolysis of your protein

Good luck!

In addition to earlier suggestions, a simple DLS (dynamic light scattering) experiment measured at a single angle will tell you if there are any large aggregates in your protein sample. If there are too many (say >0.5% by w/w) this may hinder crystallization. In contrast, a monodisperse outcome (one species namely your protein) is favorable for crystallization, although of course there are no guarantees.

Hi Rakesh,

Are you getting a reproducible homogeneous single peak in SD200 ??

In one of my project, I have one protein (~68 kDa), which is similarly not crystallizable. I observed it actually has more than two distinct domains and highly intrinsically dynamic (as observed in gel filtration), and there fore I could not be able to crystallize it.

Based on my experience I can suggest you two things:

1. Try domain crystallization (predicting domain 1st)

2. If any interacting protein is there, there is a scope that the other protein will stabilize the 1st one. ( I am trying the 2nd approach in my project).

Good Luck dear.

Thank you everyone for kind suggestions. After many trials finally I could get crystals in 2 conditions which are very similar (same pH, same PEG and concentration, salt with divalent anion). But those crystals are thin plates and could not diffract in in-house x-ray. Now I am trying to optimize to grow bigger crystals if possible.

Thanks to everyone once again.

It would be more grateful if there are any techniques to grow 3D crystals from 2D crystals?

I will conduct additive screening and seeding method. If there are other ways to get better crystals, please give me some suggestions.

I completely agree with the others: first characterize well your sample (purity, homogeneity, folding state) and optimize the buffer by some thermal melt technique (this is generally reported to have high impact). If you have enough protein and start becoming desperate, you may also consider changing the temperature of the crystallization (we succeeded at as low as 4deg but also at as high as 37deg), crystallization technique (sittinghanging drop, ev. crystallization in capillaries) or addition of nucleants (e.g. Naomi's nucleants).

If your protein is is (theoretically) charged as judged from the sequence avoid using divalent cations and use stepwise increasing NaCl (e.g 0.5M,1.0M,1.5M at some fixed protein concentration around that where you get precipitation with the 01M NaCl buffer.The divalent cations may chelate different proteins and prevent bulk growth leading to platelets instead,See our studies of protein aggregation and crystallization I have uploaded in Research Gate

Thank you everyone for your kind suggestions.

Finally, I could get the crystals of the protein but the crystal grows only in 2D as a thin plate. I did optimization, seeding and additive screening. However it only larger plates were obtained and the crystals are fragile. How can I grow crystals in 3D?

The picture of crystal is with PEG400 as additive added.

Thank you

Hi there!

I was going to write agreeing with the other people around here, but seeing as you already have some initial hits, perhaps my experience with optimization will be useful:

1. Finely tune the components of your condition. For this, perform a systematic screen. Say for example your condition is 0.1 M Buffer pH 8, 200 mM CaCl2 and 10% PEG400. In that case, I'd test from pH 7 to pH 9, from 100 mM to 500 mM CaCl2, and probably from 5% to 25% PEG400.

2. Screen for PEG length. The number behind the PEG corresponds to the average molecular weight in the PEG mix. I'd suggest you test slightly larger, and slightly shorter PEGs. So, in your case, PEG400 gave initially good results; test what happens with PEG200 to PEG600, for example.

3. Seeding! I've had a lot of success using initial crystals as seeding stock. In that case, I do both microseeding and streak seeding (owning cats is a requirement for any self respecting crystallographer, in my humble opinion).

4. Seeds are good in any condition. What I mean is that I've had it before where my initial crystals grew in condition A, I processed them into seeds, and after using them as an additive for robot assisted crystallization, my good, final crystals appeared in condition B.

5. Now for a bit of self promotion. In 2014, we published a paper in Angewandte Chemie describing the use of crown ethers as crystallization additives to help stabilize otherwise disordered lysines and hydrophobic patches. The reason we started with this project was because we noticed that, at times, short PEGs (like PEG400 or PEG200), co-crystallized with proteins in this ring-like C-shapes. Of course that's very similar to a crown ether, and indeed, we saw that, in many cases crown ethers can act as an improved version of short PEGs. I'd recommend you check out the paper (Lee, CC & Maestre-Reyna, M 2014, Angewante Chemie), and see if it may be useful for you.

Good luck!

Manuel

Thank you Manuel Maestre-Reyna for your suggestions. I had read the paper that you mentioned. I will definitely try using crown ether in next batch of crystallization. Thank you very much!!!

Hi Rakesh,

It could be that your protein too dynamic for crystallisation or it might contain a lot of unstructured parts (long loops for example). Try to run some secondary structure prediction or better CD analysis to see how well folded your protein is. If it is well folded, than you need to screen lots of conditions. You could try to lower salt in your buffer (100mM -> 20mM) if your protein survives it, or try to concentrate your protein more (until it starts to precipitate). That is usually a good starting point for crystallisation trials.

Regarding your crystals - you can definitely solve a structure from plates. Many people, including myself, have done it in the past. You just need to be very careful harvesting them, you'll probably need to screen tons of them to fish out ones which were still intact post-harvest and most likely merge datasets from isomorphous crystals.

But I'd agree with the comments above - your protein or its region is probably quite dynamic and prohibits good packing of the lattice. You can try using a software I wrote recently called "BASILIScan" to find the closest homologue to your protein whose predicted structural order is higher. You can download it for free here: www.basilisc.com and see the publication: Barski et al., BMC Genomics (2018). Good luck!

Main part of the crystal structure is carbon arrangements in 3D way which is guide lined from sequence of it. Amino acids tend to fold in such a way that it maintain carbon value (31.44%) around every atoms. Doing so the unfold-able moiety like aromatic rings may not cooperate to this value form. So net loss of force around that part which may not cooperate (non crystal). If non cooperative portions are dominant, that protein may not crystallize. So need to mutate the protein to crystallize. You may send any protein sequence which is not crystal forming. I may be able give minimum mutations to get crystal of that.