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JBS Halo-ATP Kit

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PK-101 8 nucleotides (500 nmol each) 529,52 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C

Shelf Life: 12 months after date of delivery

Applications:
Incorporation of halogenated ATP analogs into nucleotide binding enzymes for isomorphous/ anomalous phasing.

Description:
The JBS Halo-ATP Kit contains 8 halogenated nucleotide analogs as lyophilised sodium salt.

  • 2'-Iodo-dADP (#NU-109)
  • 2'-Iodo-dATP (#NU-101)
  • 2'-Bromo-dADP (#NU-110)
  • 2'-Bromo-dATP (#NU-102)
  • 8-Iodo-ADP (#NU-111)
  • 8-Iodo-ATP (#NU-113)
  • 8-Bromo-ADP (#NU-112)
  • 8-Bromo-ATP (#NU-114)

Features
Halogenated nucleotide analogs provide a straightforward method that allows rational incorporation of heavy atoms into a large number of nucleotide-binding enzymes.
ADP and ATP are brominated or iodinated at either the 8- or the 2'-position.
Bromine, with a K absorption edge of 13.4737 keV (0.9202 Å) can be used for MAD phasing and iodine is widely used for SIRAS or SAD.
It is important to keep in mind that the affinity of the phasing analogs to the protein may differ from that of non-substituted ATP-derivatives [5]. One protein may tolerate a substitution at the sugar (2') but may only weakly bind to base- (8-) substituted analogs or vice versa. Also, an excess of analog may be required in order to completely displace the bound natural ATP.
Since proteins in complex with ADP and ATP tend to show different crystallization behavior, the same may apply to proteins in complex with the respective phasing analogs. Therefore, it is worth trying the whole set of analogs as well as a range of different concentrations.

Usage
Crystal soaking as well as co-crystallization can be used to find the best binder and the highest quality crystals - from only one single 24 well tray.
Crystal soaking is the most straightforward and recommended method if you already have crystals of your protein in the apo form or already in complex with an adenosine nucleotide.

Soaking
For soaking experiments, protein crystals are transferred into a stabilizing solution drop containing the crystallization solution together with the heavy atom compound. Make sure that the stabilizing solution contains all the components of the precipitant solution in which the protein was crystallized, otherwise the crystal could start to suffer upon soaking. Concentration of the halogenated analog in the final soaking solution and soaking time is dependent on the protein under investigation. However, it is usually recommended to use a high concentration of the heavy atom compound in conjunction with a short soaking time. The recommended halogenated analog concentration in the drop is 5 mM [1]. If crystal degradation like cracking or dissolution occurs, decreasing compound concentration and extending soaking incubation time, along with a gradient soak, may be advised. Crystal cross-linking is also helpful to stabilize crystal lattice when the soaking approach is applied [2].

  • Dissolve the halogenated analog in deionized water to a concentration that is 5x your desired final concentration in the soaking solution. For instance, add 20 μl of deionized water to obtain a concentration of 25 mM halogenated analog. This stock solution is stable at -20°C for 2 weeks.

    Please note: It is possible to obtain a stock solution of the halogenated analog directly from the mother liquor where crystals have grown. Just have in mind that the solubility will be dependent on the condition in use. In this case, add 100 μl of mother liquor directly to the lyophilised in order to obtain a 5 mM soaking solution. Place a drop of 2 μl on the top of a cover slide and proceed directly to step 4.

  • Prepare a stabilizing solution, oriented on your crystallization condition, with a volume of e.g. 10 ml.

    Please note: Soaking is usually performed in a stabilizing solution, wherein the protein crystal is stable. The concentration of each component from the stabilizing solution has to be determined experimentally. As starting point we recommend the same FINAL concentrations (after addition of the heavy atom compound) as the reservoir solution where crystallization occurred. Hypotonic shocks (when concentration of stabilizing solution is lower than crystallization solution) can lead to irreversible crystal damage/dissolution while mild hypertonic shocks can cause crystal dehydration, often used to improve diffraction data [3,4].

  • Prepare 2 μl of the soaking solution on a cover slide by mixing the stabilizing solution (without the halogenated analog) with the stock solution of halogenated analog at the desired ratio, e.g. for a 5 mM analog soaking solution, mix 0.4 μl analog (25 mM) + 1.6 μl stabilizing solution.

    Remember that in this case your stabilizing solution should be 1.25x the desired final concentration.

  • Transfer the crystal into the soaking solution using a loop, or a MicroMount™ and place the cover slide on the top of a well containing the original crystallization condition.
  • Observe the crystal under a microscope to check for degradation. If no degradation occurs, incubate for one hour up to several weeks to allow analog diffusion to its binding site or the displacement reaction in the case of holo crystals. Proceed afterwards with usual crystal mounting for X-ray data collection.

Tip: Prepare the halogenated analog stock solution in deionized water ≥10x more concentrated than your desired final concentration in the soaking drop to add the analog stock solution directly to the drop containing your crystals.

Co-crystallization
If crystal soaking fails to give satisfactory results then co-crystallization is recommended. In this case, simply substitute the ATP-derivative used in your complex formation assay with the phasing-analogs and proceed with usual crystallization set up.

Selected References:
[1] Naber et al. (1995) A novel adenosine-triphos-phate analog with a heavy-atom to target the nucleotide-binding site of proteins. Protein Sci. 4:1824.
[2] Heras et al. (2005) Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Cryst. D 61:1173.
[3] Lopez-Jaramillo et al. (2002) Soaking: the effect of osmotic shock on tetragonal lysozyme crystals. Acta Cryst. D 58:209.
[4] Garman et al. (2003) Heavy-atom derivatization. Acta Cryst. D 59:1903.
[5] Gruen et al. (1999) 2'-Halo-ATP and –GTP analogues: Rational phasing tools for protein crystallography. Protein Sci. 8:2524.

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