Conference Agenda

Overview and details of the sessions of this conference. Please select a date or location to show only sessions at that day or location. Please select a single session for detailed view (with abstracts and downloads if available).

 
 
Session Overview
Session
MS-26: Solution scattering and combined techniques for biological systems, including component dynamics
Time:
Monday, 16/Aug/2021:
2:45pm - 5:10pm

Session Chair: Frank Gabel
Session Chair: Masaaki Sugiyama
Location: Club B

50 1st floor

Invited: Mitsunori Ikeguchi (Japan), Anne Martel (France)


Session Abstract

This MS will gather together interesting studies focused on clarifying aspects of complex biological systems, such as the equilibrium between association and dissociation of multiple biomacromolecules, as well as the dynamics within each biomacromolecule. Recently, structural biologists have combined several techniques in order to access such issues. In this MS, focusing on solution scattering, SAXS and SANS, and combined techniques including computational science, recent progress will be discussed.

For all abstracts of the session as prepared for Acta Crystallographica see PDF in Introduction, or individual abstracts below.


Introduction
Presentations
2:45pm - 2:50pm

Introduction to session

Frank Gabel, Masaaki Sugiyama



2:50pm - 3:20pm

Motion of a Membrane Enzyme as Seen by SANS

Waqas Javed1,2,3, Christine Ebel1, Cedric Orelle2, Jean-Michel Jault2, Anne Martel3

1IBS; Univ. Grenoble Alpes, CNRS, CEA; Grenoble, 38000; France; 2Molecular Microbiology & Structural Biochemistry (MMSB) UMR 5086; CNRS/University of Lyon; Lyon, 69000; France; 3Institut Laue Langevin, Grenoble, France

Small Angle Neutron Scattering is a low-resolution technique enabling to probe the solution structure of individual biomacromolecules possibly in complex with its partners. In particular, concerning membrane proteins, the membrane-like environment can be made invisible in order to see only the protein. Here, we combined SANS with X-ray crystallography, cryoEM, H/D exchange coupled with mass spectrometry and limited proteolysis to reveal the flexibility and ligand-induced conformational changes of the multidrug ABC transporter BmrA.

Limited proteolysis revealed an important flexibility of BmrA WT in most steps of its catalytic cycle. Cryo-EM provided high-resolution of the closed conformation by analysis of an artificially monodisperse sample, and X-ray crystallography data enabled to build homology models of other conformations, which constituted the starting point of SANS analysis. H/D X-MS pinpointed the flexible part along the transporter sequence and SANS revealed the extent of this flexibility.

Together, these techniques enable us to describe the ABC transporter cycle in term of successive conformational equilibria, a much more realistic and accurate vision of this biological process [1].

Figure 1. A: Main steps of the enzymatic cycle of ABC transporters (from [2]); B: Structural definition of these steps in solution by sequential conformational equilibria [1].

[1] Javed et al. in preparation

[2] Wannes Dermauw, Thomas Van Leeuwen, The ABC gene family in arthropods: Comparative genomics and role in insecticide transport and resistance, Insect Biochemistry and Molecular Biology, Volume 45, 2014



3:20pm - 3:50pm

MD-SAXS: Hybrid method of molecular dynamics simulations and small-angle x-ray scattering experiments

Mitsunori Ikeguchi1,2

1Yokohama City University, Yokohama, Japan; 2RIKEN, Center for Computational Science, Yokohama, Japan

Molecular dynamics (MD) is crucially important for protein functions. MD simulation is a powerful computational tool for investigating molecular dynamics of proteins in atom detail. However, due to the time-scale limitation of MD simulation, conformational samplings in MD simulation are occasionally insufficient. Thus, to validate simulation structures, the comparison of the simulation structures with experimental results is useful.

Small-angle x-ray scattering (SAXS) experiments is a powerful method to measure protein structures in solution. Although the resolution of SAXS is limited to low because of the orientational and conformational averaging, the information of protein conformations in solution can be obtained. Therefore, the comparison of simulation results with SAXS data serves to obtain the protein solution structures consistent with experiments.

We have developed a hybrid method of MD simulations and SAXS (MD-SAXS) [1­–3]. The first example of MD-SAXS applications was EcoO109I, a type II restriction endonuclease [1]. The enzyme was revealed to be substantially flexible, and the intrinsic flexibility was found to be closely related to the structural changes upon DNA binding.

Ion effects on SAXS data were investigated using MD-SAXS [2]. At a series of ion concentrations from 0 to 1 M, the MD-SAXS analysis for lysozyme was performed. The SAXS excess intensities were strongly dependent on ion concentrations. Based on the MD-SAXS, we developed a fast method to handle ion effects.

MD-SAXS was also applied to the drug target protein [4]. Vitamin D receptor (VDR) is a member of the nuclear receptor family, and functions as the control of the expression of genes through Vitamin D binding. The VDR ligand binding domain (LBD) is expected to undergo conformational changes upon agonist or antagonist binding. However, the crystal structures of VDR-LBD share a similar structure even with bound agonist or antagonist. The crystal structure of VDR-LBD in the ligand-free state has not been determined. The SAXS experiments suggest that both the ligand-free and antagonist-bound structures in solution are different from the crystal structure. Thus, the MD-SAXS analysis was performed to elucidate the solution structures of VDR-LBD in both the states. In the ligand-free and antagonist-bound state, the obtained solution structures were in good agreement with their SAXS data. Their structural features were consistent with the function of VDR.

Sampling capability of all-atom MD simulations is occasionally insufficient for very flexible and large molecules. To overcome the limitation, we developed a hybrid method of a coarse-grained MD simulations and SAXS (CG-MD-SAXS) [5]. Even in the coarse-grained models (e.g., Cα only), SAXS data were accurately reproduced from the structure models. CG-MD-SAXS was applied to the three types of nucleosomes (canonical, CENP-A, and H2A.B nucleosomes), and revealed the substantial difference in the dynamics of DNA around histones.

[1] Oroguchi, T., Hashimoto, H., Shimizu, T., Sato, M., Ikeguchi, M. (2009) Biophys. J. 96, 2808.

[2] Oroguchi, T., Ikeguchi, M. (2011) J. Chem. Phys. 134, 025102.

[3] Oroguchi, T., Ikeguchi, M. (2012) Chem. Phys. Lett. 541, 117.

[4] Anami, Y., Shimizu, N., Ekimoto, T., Egawa, D., Itoh, T., Ikeguchi, M., Yamamoto, K. (2016) J. Med. Chem. 59, 7888.

[5] Ekimoto, T., Kokabu, Y., Oroguchi, T., Ikeguchi, M. (2019) Biophys. Physicobiol. 16, 377.



3:50pm - 4:10pm

Transient complexes of the Nsp7, Nsp8 and Nsp12 in SARS-CoV-2 replication transcription complex

Greg Hura

Lawrence Berkeley National Laboratory, Berkeley, United States of America

: The RNA transcription complex (RTC) from the virus, SARS-CoV-2, is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities the RTC mechanism must also conform to a large number of imperatives including RNA over DNA base recognition, base pairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interface with error checking machinery and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8 and Nsp12 (also known as RNA dependent RNA polymerase (RdRp)). We have solved high resolution crystal structures of the Nsp7/8 complex providing insight into the interaction between the proteins. We have used small angle X-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher order complexes and with and without RNA. Using size exclusion chromatography and multi-angle light scattering coupled SAXS (SEC-MALS-SAXS) we defined which combination of components form transient or stable complexes. We used contrast matching neutron scattering to mask specific complex forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8 and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS and other biophysical techniques we provide greater insight into the RTC assembly, mechanism and potential avenues for disruption of the complex and its functions.



4:10pm - 4:30pm

The dynamics and interactions of Scs proteins from Proteus mirabilis

Andrew Whitten1, Furlong Emily2, Choudhury Hassanul2, Kurth Fabian2, Duff Anthony1, Martin Jennifer2

1Australian Nuclear Science and Technology Organisation, Lucas Heights, Australia; 2Institute for Molecular Bioscience, University of Queensland, Australia

Correct formation of disulfide bonds is critical to the folding of a wide variety of proteins. Bacterial virulence factors are one class of proteins containing disulfide bonds, thus, an approach to disarm virulent bacterial might involve shutting down the machinery involved in the formation of disulfide bonds. The suppressor of copper sensitivity (Scs) proteins form part of the disulfide bond forming machinery in bacteria, and it is hoped that determining the structure of molecules such as this may lead to the development of new classes of antibiotics. There are four Scs proteins (ScsA, B, C and D) present in numerous Gram-negative bacteria, and few have been structurally characterised. In this work we show that the ScsC protein from Proteus mirabilis is trimeric and flexible, where the high level of flexibility is afforded by a glutamine rich motif. We also show that the protein interacts with ScsB and that this interaction rigidifies the ScsC protein.



4:30pm - 4:50pm

Application of a lanthanide tag for evaluation of conformational states of a multidomain protein

Tomohide Saio1, Hiroshi Nakagawa2, Soya Hiramatsu3, Mizue Asada4, Honoka Kawamukai1,3, Toshikazu Nakamura4, Koichiro Ishimori3,5

1Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan; 2Materials Sciences Research Center, Japan Atomic Energy Agency, Tokai, Japan; 3Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan; 4Instrument Center, Institute for Molecular Science, Okazaki, Japan; 5Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan

Despite their importance in function, the conformational states and changes of proteins are often poorly understood mainly because of the lack of an efficient tool. MurD, a 47-kDa three-domain protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during its catalytic cycle are not well understood. The previous crystallographic studies have identified two major conformational states of MurD, open and closed conformations, in which the domain 3 has distinct orientations with respect to the other two domains. The conformational difference between the two crystal structures suggested that MurD can undergo drastic conformational changes in solution. However, the details about the conformational states and changes of MurD in solution coupled with the binding with the ligands or the inhibitors remained to be elucidated.

In our study, we exploited multiple biophysical methods including nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation to demonstrate evaluation of the conformational states and distribution of MurD. We exploited paramagnetic lanthanide ions that can be attached to the specific position(s) on the protein by the use of the lanthanide tags [1]. In NMR, the effects of the paramagnetic lanthanide ions are observed as pseudo-contact shifts (PCSs) that can provide long-range (< ~40 Å) distance and angular information of each of the observed nuclei in the protein [1]. The lanthanide ion was fixed on the domain 2 of MurD and PCSs were observed from the resonances derived from the domain 3. Analysis of PCSs achieved estimation of conformational states of MurD in solution and detection of the conformational changes of MurD induced by its ligands and inhibitors [2]. The paramagnetic lanthanide ions, especially gadolinium ions, can be exploited by EPR and double electron–electron resonance (DEER) measurement that provides inter-gadolinium distance and population (distance distribution) [1]. The distance distributions obtained from DEER measurement were consistent with the information derived from PCS-NMR, SAXS, and MD simulation.

Our study highlights several biophysical methods to investigate the overall conformational states of a multi-domain protein. The integrated use of these methods can be an efficient strategy to evaluate the conformational states and distribution of proteins in solution.

[1] Saio, T., Ishimori, K. (2020) Biochim. Biophys. Acta. Gen. Subj. 1864.

[2] Saio, T., Ogura, K., Kumeta, H., Kobashigawa, Y., Shimizu, K., Yokochi, M., Kodama, K., Yamaguchi, H., Tsujishita, H., Inagaki, F. (2015) Sci. Rep. 5, 16685.



4:50pm - 5:10pm

An Objective Metric to Guide Background Correction and Interepretation of Small Angle X-ray Scattering Data

Yunyun Gao1,2, Timothy R. Stachowski3, Edward H. Snell3, Thomas D. Grant3, Arwen R. Pearson1

1Institute of Nanostructure and Solid State Physics, Universität Hamburg, Hamburg, Germany; 2The Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany; 3Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA

Small-angle X-ray scattering (SAXS) experiments provide an estimation of biological macromolecule geometry on the level of domain structure. The reliability of structural inference drawn from SAXS data is dependent on the accurate measurement as well as the proper post-processing procedure. The methods improving raw data quality and gaining more information are widely explored. Among those innovations, size-exclusion chromatography small-angle X-ray solution scattering (SEC-SAXS) has become a standard method for modern bio-SAXS synchrotron light sources (Ryan et al. 2018; Brennich et al. 2016; Blanchet et al. 2015). However, the principle of data post-processing for SEC-SAXS remains rather unclear. This includes background correction and averaging of the raw data. Several statistical tools have been developed to assess solution SAXS data quality (Rambo and Tainer 2013; Franke et al. 2015). These are mostly useful for “rejecting” significantly different data points or data frames, based on the assumption that the rest of the data are close to the “truth”. But this can lead to a situation where mediocre data, for example data contaminated with radiation damage, are not correctable or simply cannot be evaluated before any interpretation is done.

To alleviate this problem, an objective metric, correction-state score (CSS) is proposed. CSS can be used to both verify the data quality and identify the optimal data correction procedure for post-processing of SEC-SAXS data. CSS can be represented as a numerical likelihood with a scale of 0 to 1. Using this objective score it is possible to quantitatively assess the “goodness” or appropriateness of a background correction for SEC-SAXS data. Under the guidance of CSS, the metadata recorded during a SEC-SAXS experiment can be used to maximise the fidelity of the post-processing as well as reduce the ambiguity in further data interpretation.

References
Blanchet, C.E., Spilotros, A., Schwemmer, F., et al. 2015. Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY). Journal of Applied Crystallography 48(Pt 2), pp. 431–443.
Brennich, M.E., Kieffer, J., Bonamis, G., et al. 2016. Online data analysis at the ESRF bioSAXS beamline, BM29. Journal of Applied Crystallography 49(1), pp. 203–212.
Franke, D., Jeffries, C.M. and Svergun, D.I. 2015. Correlation Map, a goodness-of-fit test for one-dimensional X-ray scattering spectra. Nature Methods 12(5), pp. 419–422.
Rambo, R.P. and Tainer, J.A. 2013. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496(7446), pp. 477–481.
Ryan, T.M., Trewhella, J., Murphy, J.M., et al. 2018. An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis. Journal of Applied Crystallography 51(1), pp. 97–111.