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Phasing of Nucleic Acid Crystal Structures with Phosphorus Anomalous Signal

Applicant Olieric Vincent
Number 129584
Funding scheme Project funding (Div. I-III)
Research institution Paul Scherrer Institut
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Inorganic Chemistry
Start/End 01.11.2010 - 31.10.2013
Approved amount 151'992.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Biophysics

Keywords (8)

crystallography; structure; phasing; phosphorus; nucleic acids; RNA; riboswitch; structures

Lay Summary (English)

Lead
Lay summary
The Ph.D. thesis "Phasing of Nucleic Acid Crystal Structures with Phosphorus Anomalous Signal" at the Paul Scherrer Institut (Villigen PSI, Switzerland) aims at developing new methods in crystallography for the 3D structure determination of nucleic acid (DNA/RNA) and nucleic acid/protein complexes. Such work is of particular importance for structural biologists working both in academia and in the pharmaceutical industry since structures might be solved more easily and faster. With the growing number of biologically important nucleic acid sequences, there is indeed a need to develop a general and fast strategy to ease their crystal structure determination. Initial experiments at the Swiss Light Source synchrotron (SLS at Paul Scherrer Institut) have shown that medium-size RNA structures can be determined using the "anomalous" signal of phosphorus atoms only (phosphorus is a component of RNA). We now want to further extend this method, called Phosphorus-Single Anomalous Diffraction (P-SAD), to tackle larger structures. To attain this goal, the Ph.D. student will work on the methodological development of crystallography in close collaboration with Dr. Philippe Dumas at the University of Strasbourg, France. To prove the validity of the developed method, we plan to solve a large nucleic acid structure. This will be done in collaboration with Pr. Dr. Roland Sigel (also Ph.D. supervisor) at the University of Zürich, where the work will focus on the structure of a complex (btuB riboswitch RNA / metabolite B12) involved in gene regulation expression which could serve as a starting point for structure-based drug design of new antibacterials. Covering a wide range of biochemical and structural biology techniques, this thesis project represents a good oportunity for a Ph.D. student. The work will also highly benefit of the latest instrumentation developments at the SLS macromolecular crystallography beamlines, among them are the crystallization facility, the novel hybrid-pixel X-ray detector and a new multi-axes goniometer. The Ph.D. student, Guanya Peng, has been recruited and has started on November, 2nd, 2010. He works at the SLS under the supervision of Dr. Vincent Olieric (main proposer), beamline scientist and Dr. Meitian Wang, group leader of the macromolecular crystallography group.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Name Institute

Collaboration

Group / person Country
Types of collaboration
Institut de Biologie Moléculaire et Cellulaire France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Zürich Universität Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
27th Rhine-Knee Regional Meeting on Biocrystallography Talk given at a conference 25.09.2013 Schluchsee, Germany Peng Guanya;
4th Winter School on soft X-rays in Macromolecular Crystallography Poster 06.02.2012 ESRF Grenoble, France Peng Guanya;
Extended Wavelength X-ray Crystallography Workshop at the 2011 APS/CNM/EMC Users Meeting Talk given at a conference 04.05.2011 Advanced Photon Source, USA, United States of America Wang Meitian;


Self-organised

Title Date Place

Associated projects

Number Title Start Funding scheme
182369 Development of native-SAD phasing for membrane protein structure determination 01.07.2019 Project funding (Div. I-III)

Abstract

With the growing number of biologically important nucleic acid sequences, there is a need to develop a general and fast strategy to ease their crystal structure determination. Technically, the simplest approach to solving the so-called "phase problem" in macromolecular crystallography is the use of single-wavelength anomalous dispersion (SAD) signal. For proteins, Sulfur-SAD phasing (S-SAD) has become an appealing method especially for high-throughput structural projects since the protein itself or the crystal do not require any modification (selenomethionine incorporation or heavy atoms derivatization). Nucleic acids do not contain sulfur (except thio-modified nucleoside residues), but a considerable amount of phosphorus is present naturally in all DNA / RNA (Desoxy- / RiboNucleic Acids) and can therefore be used for phasing in a method called Phosphor-SAD or P-SAD. However, owing to the fact that the anomalous signal constitutes only a small fraction of the total crystal scattering and that SAD phasing requires an accurate measurement of reflection intensities, this method has been so far of limited use. The purpose of this Ph.D. project is to overcome those difficulties by improving both data collection protocols and phasing procedures and to later apply the developed tools for the determination of an important and challenging nucleic acid structure. At Paul Scherrer Institut, we have recently solved the structure of the Sarcin Ricin Loop RNA (27 nucleotides) using the anomalous signal from phosphorus only, demonstrating that the P-SAD method is perfectly valid for medium-size RNA molecules. To further extend the method to larger nucleic acids, the Ph.D. student will pursue three main goals. The first one is to delineate the limits of the method in terms of data quality, redundancy and energy used for data collection. This will be done with simulated data as well as data collected on various RNA molecules provided by the group of Dr. Philippe Dumas (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France). The second goal is to improve the methods to locate phosphorus atoms for successful phasing. Progress on this side will allow to expand the method to large-size RNAs for which the upper resolution limit is often within 2.5 - 3.0 ?. This represents a very challenging case for P-SAD only but we believe that successful phasing can be achieved by combining P-SAD with molecular replacement using small RNA fragments as search model and by using the general knowledge of nucleic acids structure. The ultimate goal is to prove the validity of the method by solving a large nucleic acid structure. In collaboration with the group of Pr. Roland Sigel (University of Zürich, Switzerland) who will also act as Ph.D. supervisor, we will work on the structure determination of the btuB riboswitch RNA (202 nucleotides) in complex with its metabolite B12. Riboswitches RNA are uniquely capable of controlling the expression of genetic information and can thus serve as antibacterial drug target. Revealing the specificity of such a complex is therefore of prime interest in the drug discovery process.The Ph.D student will work at the Swiss Light Source (SLS at Paul Scherrer Institut, Villigen PSI, Switzerland) under the supervision of Dr. Vincent Olieric (main proposer) and Dr. Meitian Wang, both are beamline scientists in the group of Dr. Clemens Schulze-Briese. The work will highly benefit of the latest instrumentation developments at the SLS macromolecular crystallography beamlines (among them are the crystallization facility, the novel hybrid-pixel X-ray detector and a new multi-axes goniometer). Moreover, the Ph.D. student will profit of the expertise found in the group of Dr. Philippe Dumas for the theoretical part of the work and in the group of Pr. Roland Sigel for the preparation of large RNA molecules.
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