Moody Lab FAQ

Using TELSAM Fusion Crystallography (TFC)

Q: What proteins have been crystallized using TFC so far?
A: The von Willebrand factor A domain of human capillary morphogenesis gene II, PDB ID: 8FT8.
The ubiquitin-associated domain of human thirty-eight negative kinase 1, PDB ID: 7TDY.
An arbitrarily chosen DARPin, PDB ID: 9DB5.
SARS Cov2 nsp14 N7-MethylTransferase domain, PDB ID: 7TW9.

Q: What kinds of proteins is TFC good for?
A: Currently, monomeric proteins up to around 50 kDa are expected to crystallize well as TELSAM fusions.

Q: How do I design a TELSAM fusion?
A: Visit our tutorial page.

Q: What are the current best practices for using TFC?
A:
1) Use the 1TEL version of TELSAM, which displays a copy of the target protein at the C-terminus of every TELSAM subunit. In a controlled study, CMG2.vWa and TNK1.UBA target proteins formed far superior crystals when fused to 1TEL than when fused to other forms of TELSAM (e.g. 2TEL, 3TEL) that space target proteins further apart (manuscript in preparation).

2) Engineer the shortest possible linker between the 1TEL C-terminus and the protein of interest. https://doi.org/10.1016/j.str.2023.09.001

3) Carefully consider the conformational flexibility of the linker. Proline, threonine, and valine work well for this.22 Some target proteins perform better with flexible glycine linkers or semi-rigid α-helical linkers. When flexible, semi-flexible, and α-helical linkers of varying lengths were tested for their ability to crystallize a DARPin, the α-helical linkers performed best. Conversely, when these classes of linkers were tested with a TNK1.UBA construct, the flexible linkers prevailed (manuscript in preparation).
https://doi.org/10.1038/s41594-022-00828-1
https://doi.org/10.1107/S2059798323007246

4) Use a cleavable purification tag at the 1TEL N-terminus. In a controlled study using flexible and semi-flexible linkers, both DARPin and TNK1.UBA constructs formed far superior crystals if the 10xHis tag was cleaved prior to crystallization (manuscripts in preparation). We have had good success using a 10xHis-SUMO tag and cleaving it using SUMO protease. Using instead a semi-rigid α-helical linker does not require the removal of small (e.g. 10x Histidine) purification tags because with this linker the fused target protein cannot fold back to dock against its host TELSAM polymer, a conclusion validated by obtaining high-resolution (1.8 Å) crystals of a DARPin fused to 1TEL using an α-helical linker (manuscript in preparation).
https://doi.org/10.1098/rsob.210271

5) Purify the 1TEL–target protein fusion at pH 8.8–9.5 using affinity chromatography, purification tag cleavage using minimal protease, and size exclusion chromatography (SEC), followed by screening 300-400 sparse crystallization conditions at both 1 mg/mL and 10–20 mg/mL protein.
https://doi.org/10.1098/rsob.210271

Q: What do I do if my TELSAM fusion construct is insoluble after cell lysis?
A: Try a higher buffer pH, up to 9.5
Try a second pH trigger (L96E, by the original gene numbering in Uniprot: P41212, ETV6_HUMAN)
Try screening protein solubility additives (Hampton Research part number HR2-072, for example)

Q: What do I do if my TELSAM fusion construct starts precipitating during purification?
A: Try a higher buffer pH, up to 9.5
Try a second pH trigger (L96E, by the original gene numbering in Uniprot: P41212, ETV6_HUMAN)
Try screening protein solubility additives (Hampton Research part number HR2-072, for example)

Q: What do I do if my TELSAM fusion construct doesn't crystallize?
A: Try a different character of linker (GlyGly vs. ProAla vs. a direct helical fusion)
Reach out to us!

Q: What do I do if my TELSAM fusion construct only forms very small crystals?
A: Try optimizing your crystallization conditions
Try micro-seeding into sparse screening conditions

Q: What do I do if my TELSAM fusion crystals don't diffract?
A: Try a different character of linker (GlyGly vs. ProAla vs. a direct helical fusion)
Try diffracting a different crystal form
Try optimizing your crystallization conditions
Try micro-seeding into sparse screening conditions

Q: What do I do if my TELSAM fusion construct diffracts to only poor resolution?
A: Try a different character of linker (GlyGly vs. ProAla vs. a direct helical fusion)
Try diffracting a different crystal form
Try optimizing your crystallization conditions
Try micro-seeding into sparse screening conditions

Q: What do I do if my TELSAM fusion construct diffracts well but molecular replacement fails or gives an unrealistic solution?
A: Try a different character of linker (GlyGly vs. ProAla vs. a direct helical fusion)
Try diffracting a different crystal form
Try optimizing your crystallization conditions
Try micro-seeding into sparse screening conditions

Q: How do I know if my crystals exhibit TELSAM polymer flipping or broken lattice periodicity?
A: Indexing gives a P6122 space group (polymer flipping)
Phenix Xtriage or similar software detect a significant translational pseudosymmetry (broken periodicity)