Hi Sherif, it depends. Regarding the 'current' protein :

- What is the general outcome of the screens tried? Is there mostly precipitation and if so what type of precipitation? Or do drops stay mostly clear?

- Can you improve the purity of the protein further (e.g. extra chromatography technique)?

- Did you check the stability of the protein preparation in the current buffer, and is this the optimal buffer?

- Did you check the aggregation state of the protein in solution (e.g. by dynamic light scattering)?

- Did you do the crystalization screening at different temperatures?

Options :

- You may perform a more systematic screening by preparing your own crystallization matrices (e.g. concentration series of PEGs or salts or oganic solvents at different pH's).

If this doesn't work out you could :

- change the construct (regarding tags : different tag type, N-/C-terminal)

- try a homologue

The list may not be complete but gives you some ideas hopefully.

Best, Tjaard

Hi Sherif,

First thing you have to check would be how easy your protein may crystallize.

http://xtalpred.godziklab.org/XtalPred-cgi/xtal.pl

this server will give you complete picture if it does not, than why.

One thing you would like to check, if your protein has signal peptide (it has to be removed). If the server predicts flexible N-term or C-term (you can cut them, which means a new construct).

If your protein has to have co-factor (ligand, metal, FAD, NAD ....) incubation before the crystallization will increase your chances.

Some proteins do not fold properly without a partner. Or they are not stable without them. You can try to co-crystalize with the partner.

If the model of similar structure exists in PDB, check if domains are moving. And what could "fix" their positions (ligand, another protein).

If your protein is a membrane protein you have to use special screens with detergents. And possibility to crystalize membrane protein is much lower than other proteins.

If size of your protein is too small, maybe NMR would be a better choice.

If the size of your protein (or complex) is too big, maybe CryoEM would be a better choice.

Good luck!

George.

Hi Sherif,

I don’t know the property of your target protein, so I can only give you some basic advice.

1. Try as many screening conditions as possible. After all, growing the crystal is a matter of luck. Focus on the conditions of precipitation, which is a good omen because I think the crystal is regular precipitation;

2. Understand the nature of your target protein and analyze which buffer system is more suitable for your protein, Tris/MES/Hepes...? Does it require a special operation, such as exposure to air for a while during crystallization...?

3. Maybe the current expression and purification conditions cannot guarantee that your protein used for crystallization is in a stable state, so it is recommended that you try more constructions and expression systems;

4. If none of the above attempts can solve your problem, I suggest you consider other structural biology methods that don’t require crystallization. Recommend an article for you, maybe can help you choose the experimental method to determine the protein structure. https://www.creative-biostructure.com/comparison-of-crystallography-nmr-and-em_6.htm

Good luck!