Crystallophore Experimental Phasing Agents

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 trials).

These are:

LuXo4-01-P as PHASING AGENT (LuXo4-01/0.6mg to use) – Sold Per Microtube Unit with 0.6mg in it
LuXo4-01-P is fully compatible with conventional phasing methods. LIII absorption edge of Lu-Xo4, at 1.34 Å, is easily accessed with most synchrotron-radiation sources leading to an anomalous contribution of about 30 e-. This contribution is also important (10 e-) on a fixed-wavelength (1 Å) beam line allowing non-optimized SAD phasing

TbXo4-02-P as PHASING AGENT (TbXo4-02/0.66 mg to use) – Sold Per Microtube with 0.66 mg in it
TbXo4-02-P is fully compatible with conventional phasing methods. It is a straightforward PHASING AGENT thanks to huge anomalous properties of f-block elements (@7.51 KeV) facilitating crystal structure determination using SAD or MAD methods.

A major advantage is that LuXo4-01-P and TbXo4-02-P overcomes the tedious and time-consuming work of seleno-methionine labeling or of heavy atom incorporation. Prior to data collection, a rapid soaking of crystals (from 30 sec to 5 min) in a concentrated Crystallophore solution (100mM) is done in their respective mother liquor supplemented with a cryo-protectant. (LuXo4-01-P and TbXo4-02-P are supplied in 0.5 ml microtubes having a conical bottom and screw cap under Argon.)

Summary

When used as phasing agents (for soaking protein native crystals or co-crystals obtained with 10mM of the same Crystallophore), a high concentration of the Crystallophore is required (100mM : 10 times more than for nucleation) : according to their respective molar mass, the packaging allow you to prepare 10 µl of soaking liquor. The phasing properties are due to the Lanthanide atom “trapped” in agents: Tb=Terbium and Lu=Lutetium. Each one has its own characteristics; you choose the one that is most suited to our needs.

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Product Information

The quantities of each of these products are different (0,6 mg and 0,66 mg) because of their respective molar mass.

Polyvalan Crystallophore Phasing Protocol

Instructions for LuXo4-01-P and TbXo4-02-P as PHASING AGENT

Methods of use:

  1. A short centrifugation of the microtube allows the powder* to form a pellet.
  2. The pellet is then re-suspended with 10 µl of mother liquor containing a cryo-protectant to reach a final Crystallophore concentration of 100 mM.
  3. The solution is centrifuged at 10000 rpm for 2 minutes.
  4. A drop of 1µl is deposited on a coverslip
  5. Crystals are harvested and soaked in the drop containing Crystallophore and the mother liquor with cryo-protectant (no back soaking)
  6. Crystals are flash frozen in liquid nitrogen
    NB: Phasing with Crystallophore (100mM) is also recommended with co-crystals obtained with 10mM of the same Crystallophore.

*can also be a light brown oil: the aspect of the product can be distorted by an effect of temperature or transport, but it does not change its efficiency.

To fully exploit the anomalous contribution of lutetium or terbium, a X-ray fluorescence scan can be performed to precisely define the peak and the inflection wavelengths of the lanthanide LIII absorption peak and to set the energy of the X-ray beam.