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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The quantities of each of these products are different (0,6 mg and 0,66 mg) because of their respective molar mass.
Instructions for LuXo4-01-N and TbXo4-02-N as a PRE-SCREENING AGENTS.
Methods of use:
- A short centrifugation of the Xo4-N microtube allows the powder* to form a pellet.
- The pellet is then re-suspended with 25 µl of protein solution to reach a final Xo4 concentration of 10 mM.
- Mix it (the protein solution may turn cloudy without affecting the subsequent screening. Check for nano/micro crystals)
- The solution shall be immediately used for crystallization.
- 2' It’s also possible to play with the Xo4 concentration, as hits have already been observed from 2 mM and up to 50 mM and more: by forming the drop through the mixing of 1 vol of protein solution + 1 vol of Xo4 solution + 1 vol of precipitant (in that order = Xo4 first). In the case of a cloudy solution after addition of the protein solution, repeat the process by dissolving first the crystallophore powder with a solution of NaHCO3 10mM at pH 7
- 3' Use it immediately for your screening trials by doing a 1+1+1 drop (in that order: protein solution + Xo4 solution + precipitant solution)
Use mother liquor or aliquots of Xo4 rapidly and store them frozen at -80°C until further use.
Depending on the protein, different screens can be used. In order to get as many exploitable hits as possible, the optimal crystallization experiment would combine screening of conditions of the protein both in native conditions and in presence of Xo4 (i.e.: two drops shall be made, without and with).
*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.