XRAD (Crystallographic Radiation Studies)

Project leaders

Alberto Cassetta, Sandor Brockhauser

Agreement

UNGHERIA - HAS (MTA) - Accademia Ungherese delle Scienze

Call

CNR/HAS (MTA) 2016-2018

Department

Chemical sciences and materials technology

Thematic area

Chemical sciences and materials technology

Status of the project

New

Research proposal

Background: Single crystal X-ray diffraction has established as the method of choice in macromolecular structure determination at atomic level. Despite the rapid evolution of both methods and instrumentations, the so-called "phase problem", the inference of the phases of the diffracted waves, may still be a major hurdle when determining the crystal structure of a macromolecule. Single wavelength Anomalous Diffraction (SAD) exploiting the weak anomalous scattering due to sulfur (S-SAD) or phosphorus, constitutive elements of proteins or nucleic acids, has emerged as one of the most promising methods in macromolecular crystallographic phasing [1]. The acquisition of a sensible signal from weak anomalous scatterers, such as S or P, primarily requires the use of an intense X-ray source such as a third-generation synchrotron, operating at wavelength in the range of 1.7-2.5 Å. A special care in the data acquisition protocols is also required. In order to have a sensible anomalous signal the highest number of diffraction spots has to be collected before the crystal being irreversibly damaged from the synchrotron beam (radiation damage). Often the acquisition of data from multiple crystals or from a single crystal oriented in different ways is also required. The combined use of a k-goniometer and an efficient data collection strategy, allowing for flexible alignment of the crystal sample, makes possible the reduction of both the overall exposure time and the radiation damage effects. K-goniometers and intelligent crystal re-orienting procedures are nowadays recognized as essential parts of successful S-SAD phasing protocols.

Importance of the proposal: The XRD1 beamline is a joint collaboration between CNR - Istituto di Cristallografia (CNR-IC) and Sincrotrone Trieste, which operates in the Hard X-ray region at the ELETTRA Synchrotron (Trieste). XRD1 exploits the intense X-ray beam produced by a Wiggler insertion device and is equipped with state-of-the-art optical components, a fast-readout detector (Pilatus) and a Huber k-goniometer. Moreover, a sample-changer for semi-automatic crystal mounting and centering has been locally developed and implemented, for high-throughput operations. XRD1 allows for conventional single-wavelength data collection, as well as wavelength-optimized SAD or MAD data collections. XRD1 is open to a wide community of users, especially from Italy and Est-European countries and it is at the heart of the Structural Biology and Material Science activities at CNR-IC in Trieste [2].
Considered the intrinsic continuous spectrum of the Wigglers and the critical energy that characterizes the one operating at XRD1, the beamline, which operates in the Wavelength range of 0.6-3.0 Å, is especially suited for S-SAD experiments. While XRD1 is equipped with a fully automated k-goniometer, high-level data-collection strategies are not currently implemented into the beamline control system. In order to fully exploit its potentiality in long-wavelength phasing XRD1 urges sophisticated multi-axis re-orientation software coupled to intelligent data-collection strategy software. Moreover, some hardware upgrade in reducing the diffused radiation from both the air and the sample, particularly strong at long wavelength, may be needed. The implementation of optimized protocols for data collection will result in an overall improvement in the quality of data from XRD1, but we expect a drastic improvement for all those cases where the macromolecular crystals exhibit a weak anomalous signal.

Research scopes: The aim of the present project is twofold: the development and implementation of robust and flexible data collection protocols, which will be implemented at XRD1, and their application to hard-to-solve macromolecular crystal structures.
The first aspect of the project strongly relies on the STAC (Strategy for Aligned Software) software, developed by Dr. Brockhauser and currently in use in many X-ray diffraction beamlines at European Synchrotrons [3]. This part of the project will require a strong interaction between the Italian and the Hungarian research groups, considered the specific setup of XRD1. Furthermore, new and more efficient solutions may be experimented with the aim of reducing the radiation damage, increasing data collection efficiency and finally increase the strength of the anomalous signal for S-SAD applications.
The second aspect of the program will deal with data collection and experimental phasing from macromolecular crystals which are recalcitrant to structure solution or which could be solved exploiting the S atoms in the native structures. While in this second aspect of the project the goodness of the new data collection strategies will be tested, the structure of molecules relevant in biology and medicine, which are essential for the understanding of the mechanisms underlying life, are expected to be elucidated.

Advantages of the collaborations: Once improved by STAC implementation, XRD1 will be an outstanding instrument for long wavelength crystallographic phasing experiments, with general advantages for the user community but especially for the Italian and Hungarian research groups. The vast expertise in data collection optimization and in radiation-damage reduction of the Hungarian group will perfectly match the specific expertise on XRD1 of the CNR staff. The research group from ELETTRA Synchrotron will be also involved in the development of the project. The Hungarian group has established a Crystallographic facility in Szeged especially aimed at macromolecular crystallography and the CNR group in Trieste is involved in structural biology projects. A collaboration between the two groups in Structural Biology is planned, eventually exploiting the facilities available at ELETTRA and in Szeged.

[1] Weinart et al. Nature Methods (2015) 12, 131
[2] Lausi et al. Eur. Phys. J. Plus (2015) 130, 43
[3] Brockhauser et al. Acta Cryst (2013) D69, 1241

Research goals

The final deliverables of the present project are:
1) The development of new methodologies for single-crystal X-ray diffraction data-collection. The new methodologies aim at reducing the radiation damage problem and increasing the significance level of the anomalous scattering components, from crystal which are poor anomalous scatterers.
2) The implementation of the new methodologies developed into the XRD1 Beamline Control System. Together with some improvement in the XRD1 hardware, the implementation of more efficient data-collection protocols will make XRD1 especially suited for long-wavelength SAD data collection.
3) Solve new macromolecular structure exploiting the newly developed methods implemented at XRD1 or in Szeged.
We intend to reach the project objectives by implementing the software developed from Dr. Brockhauser, at the XRD1 beamline at ELETTRA, in order to reduce the radiation damage effects. Proper alignment protocols will be of the greatest importance to maximize the anomalous signal but also to obtain highest data completeness. Further improvement in both the strategies of data collection and air-scattering reduction are also a project objective, with the aim of increase the significance level of the anomalous signal.
As a part of the test procedure, we intend to solve, by experimental phasing, novel crystal structures whose structure are currently not known; some of them, which are recalcitrant to be solved, are already under study in our laboratories.

Last update: 13/05/2024