Protein crystal structure determination with the crystallophore, a nucleating and phasing agent.

Sylvain Engilberge,Seigo Shima,Olivier Maury,Tristan Wagner,Eric Girard,Bruno Franzetti,Cécile Breyton,Gianluca Santoni,Francois Riobé

doi:10.1107/s1600576719006381

Abstract

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome by the crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating and phasing properties. This article presents examples of new crystallization conditions induced by the presence of Tb-Xo4. These new crystalline forms bypass crystal defects often encountered by crystallographers, such as low-resolution diffracting samples or crystals with twinning. Thanks to Tb-Xo4's high phasing power, the structure determination process is greatly facilitated and can be extended to serial crystallography approaches.

Highlights

Crystallography is the method of choice for obtaining atomic scale structural information on biological macromolecules and has contributed significantly to the development of structural biology, as shown by the number of structures present in the Protein Data Bank (PDB; https://www.rcsb.org/)
Within the framework of our first study (Engilberge et al, 2017), we observed that samples obtained by cocrystallization with 10 mM Tb-Xo4 were not always sufficiently derivatized to ensure a successful phasing, even when the crystallophore induced a clear effect on the crystallization process
The following protocol was applied: (i) crystals were grown in the presence of 10 mM Tb-Xo4; (ii) for data collection, crystals were soaked for 2 min in a concentrated solution of Tb-Xo4 by adding 2 ml of cryo-solution containing 100 mM Tb-Xo4 directly onto the crystallization drop; (iii) crystals were harvested and immediately cryo-frozen in liquid nitrogen

Summary

Introduction

Crystallography is the method of choice for obtaining atomic scale structural information on biological macromolecules and has contributed significantly to the development of structural biology, as shown by the number of structures present in the Protein Data Bank (PDB; https://www.rcsb.org/). In a non-exhaustive way, we can quote (i) automation of the crystallogenesis process by means of pipetting robots, allowing the use of ever smaller volumes of biological material (nanodroplet crystallization) (Santarsiero et al, 2002; Brown et al, 2003); (ii) rationalization of these conditions via the numerous crystallization kits available on the market; (iii) synchrotron light sources with their tuneable, micro (nano)-focus, automated beamlines and more recently X-ray free-electron laser (XFEL) sources; (iv) phasing methods exploiting anomalous scattering (Hendrickson, 2014), associated, for example, with selenomethionine labelling (Doublie, 1997) or exploitation of the intrinsic sulfur anomalous signal (Liu et al, 2012; Weinert et al, 2014) In this respect, the structural genomics projects initiated in the 2000s have strongly contributed to these technological leaps. Outcome statistics show that the success rates of the major steps in the crystal structure determination

Results

Discussion

Conclusion