A new class of lanthanide complexes to obtain high-phasing-power heavy-atom derivatives for macromolecular crystallography

Abstract

Because of their intense white lines and large f HH values, lanthanide atoms are of great interest for solving structures of biological macromolecules using single-wavelength anomalous diffraction (SAD) or multiple-wavelength anomalous diffraction (MAD) methods. In this work, a series of seven gadolinium complexes are described which provide excellent derivatives for anomalous diffraction experiments in biological systems. These highly soluble lanthanide complexes can easily be introduced into protein crystals either by soaking or by co-crystallization, without signi®cantly affecting the crystallization conditions, by employing highly concentrated complex solutions (9100 mM). De novo phasing by the SAD method was carried out with several proteins of known as well as previously unknown structures by employing this new class of heavy-atom compounds. Diffraction data were collected either with a laboratory source, making use of the high anomalous signal (f HH = 12 e À ) of gadolinium with Cu K radiation, or with synchrotron radiation at the peak of the gadolinium L III absorption edge, which exhibits a strong white line (! = 1.711 A Ê , f HH = 28 e À ). Using one of these gadolinium complexes, Gd-HPDO3A, the structure of a bacterial chimeric ornithine carbamoyl transferase, OTCase3630, a dodecameric protein of 450 kDa, was determined. Employed with the SAD method, these seven complexes could be of particular interest for high-throughput macromolecular crystallography.

Structure of the Mediator head module

“…Crystals were collected and transferred in a stepwise manner to the final cryo-solution (50 mM MES, pH 6.0, 100% saturated lithium sulphate) and flashfrozen in liquid nitrogen. For structure solution native crystals were derivatized with Ta 6 Br 12 (refs 41-43) (Proteros biostructures) and Yb-DTPA-BMA 44 (NatXray). Ta 6 Br 12 was added directly to the crystallization drop at 2 mM for 1 h. Yb-DTPA-BMA was added to the final cryo-solution at 100 mM for 10 min and back-soaked 10 s before freezing.…”

A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana

“…The structure of VDE cd was solved by the single-wavelength anomalous diffraction method using a protein crystal grown at pH 5.0 and soaked in a Gadolinium solution (Girard et al, 2003). Subsequently, the structure of VDE cd was solved by molecular replacement using a crystal grown at pH 7.…”

“…The structure of the acidic form of the protein was solved by the single-wavelength anomalous diffraction method using crystals soaked for 2 h in the same solution that serves to grow them, but supplemented with 100 mM GdHPDO3, a neutral Gadolinium complex (Girard et al, 2003). Diffraction data up to 2.5 Å (peak) or 2.0 Å (remote) resolution were collected with the FIP beamline (ESRF), reduced using Mosflm (Leslie, 1993) and scaled using Scala (Evans, 1997) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994).…”

The prominent role of resonant elastic scattering for solving the X-ray structure of macromolecules

“…However, incorporation of lanthanides by using lanthanide salts often damages protein crystals, owing to the preferred nine-based coordination of lanthanide ions. One solution to overcome this problem is to use lanthanide complexes made of a ligand that surrounds the lanthanide ions as a cage [14,15]. These complexes can be easily introduced in protein crystals and turn out to be efficient vehicles for phasing using anomalous dispersion, in particular for large macromolecular assemblies (for a short overview of protein structures determined using these lanthanide complexes, see [13]).…”