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3 NUCLEATING AGENTS

Each Polyvalan Crystallophore products can be used both as pre-screening agents and phasing agents.

The difference is how they are used depends on how they are packaged.

You have your choice of two Crystallophore Pre-Screening Agents. Both are mainly adapted to soluble proteins and complexes. Their new packaging is now adapted for limited amounts of protein (25 µL). Crystallophore Pre-Screening Agents are sold in packs of 5 vials.

When used as pre-screening agents (for getting protein crystals), the aim is to enable users to limit their costly protein consumption. This is why the pre-screening agents are provided in small packaging adapted to 25 µL protein solution. The quantities of products are different (0,15mg and 0,17mg) because of their respective molar mass. We consider LuXo4-01 and TbXo4 v1/TbXo4-02 as complementary nucleating agents.

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3 PHASING AGENTS

Each Polyvalan Crystallophore products can be used both as pre-screening agents and phasing agents.

The difference is how they are used depends on how they are packaged.

You have your choice between two Crystallophore Experimental Phasing Agents both designed to improve phasing properties. Crystallophore Phasing Agents are sold by unit (1 vial): 10μL of soaking solution (≈ 10 soaks).

When used as experimental phasing agents (for soaking protein crystals), a high concentration of the Crystallophore is required (100mM : 10 times more than for nucleation) : according to their respective molar mass, the packaging allow users to prepare 10 µl of soaking liquor (≈ up to 10 soaks). The phasing properties are due to the Lanthanide atom “trapped” in the agents: Tb=Terbium and Lu=Lutetium. Each one has its own characteristics: the user can go to the one that seems the most suitable to their needs.

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Browse our technotes to choose the right product/protocol

Tb-Xo4 technotes

Tb-Xo4 acts as a strong Nucleating Agent providing new crystallisation conditions and generally leading to the formation of bigger Tb-Xo4 derivatized crystals, ready-to-use for X-ray diffraction. Under UV irradiation, it emits in the green (Luminescent) enabling very easy detection of the Tb-Xo4 derivatized crystal in crystallization drops. It is also a straightforward Phasing Agent facilitating crystal structure determination using SAD or MAD methods, due to the exceptional anomalous properties of f-block elements.

TbXo4-02 User GuideTbXo4-02 MSDSTbXo4 v1 PDB 7MT CIF

LuXo4-01 technotes

Lutetium based Crystallophore LuXo4-01-N does not only provide new crystallization conditions but it also improves the quality of the obtained crystals. LuXo4-01 presents complementary nucleation enhancement to TbXo4. Both are mainly adapted to soluble proteins and complexes. This complex is not luminescent and has been designed to improve phasing properties : its LIII absorption edge, at 1.34Å, is easily accessed with most synchrotron-radiation sources leading to an anomalous contribution of about 30e-. This contribution is also important (10e-) on a fixed-wavelength (1Å) beamline allowing non-optimized SAD phasing.

LuXo4-01 User GuideLuXo4-01 MSDS

FAQ

What is the usual final concentration of Xo4 in the protein solution ?

Xo4 should be dissolved by mixing it with protein solution to reach a final concentration of 10 mM. Then the crystallization experiments should be set up by mixing a volume of this « protein + Xo4 solution » with an equal volume of precipitant solution.

What is the range of final concentrations of Xo4 in the sample ?

The proposed 10 mg/ml concentration is the optimal one. When hit is obtained, it’s possible to conduct an optimization by playing with the protein solution volume used in order to vary the Xo4 concentration from 1 to 10 mM.

Are there any problems using additives such as phosphate, EDTA or EGTA ?

There should be some competition with EDTA or EGTA to chelate the Lanthanide but we don’t know what will be the cinetic for a potential Lanthanide decorporation that could affect Xo4 efficiency. If EDTA or EGTA concentration is low (< 2 mM), this could only affect up to 20% of Xo4 complexes : the unaffected 8 mM Ln-Xo4 are then still falling inside its efficiency range.

Is there any limitation on pH or buffer composition or possible ligands in co crystallization ?

Xo4 has been designed in order to be stable and highly soluble in aqueous medium and consequently fully compatible with high throughput screening. It is stable in all crystallisation conditions that have been tested ; please note that in some cases the presence of phosphate can be detrimental to the efficiency of Xo4.

When mixing the protein with Xo4, the solution may turn milky without affecting the subsequent screening. Check it for nano/micro crystals

What are the preferred binding sites or amino acids for Tb-Xo4 ?

There is direct interaction between protein negatively charged residues but not only.

Are some groups of enzymes inhibited by Xo4 ?

It has never been observed but we cannot exclude it for very specific proteins.