MS04-P08 Liquid Dense Clusters: Intermediates between Nanocrystals and Single Particles Hevila Brognaro (Center for Free Electron Laser Science. Deutsches Elektronen-Synchrotron (DESY)., Hamburg, Germany) Sven Falke (Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Institute for Biochemistry and Molecular Biology, University of Hamburg,, Hamburg, Germany) Robin Schubert (Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Institute for Biochemistry and Molecular Biology, University of Hamburg, , Hamburg, Germany) Christian Betzel (Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Institute for Biochemistry and Molecular Biology, University of Hamburg, , Hamburg, Germany)email: hevila.brognaro@desy.deLiquid-dense clusters (LDCs) are formed by liquid-liquid phase separation, which is a process known for a long time in vitro as well as in vivo affecting a variety of nucleic acids, protein functions and metabolic pathways. Insights in the formation and dynamics of liquid dense clusters will open also new option to optimize protein crystal growth [1].
Nonetheless, the knowledge about structural and dynamic properties of macromolecule LDCs of proteins and different nucleic acids remains by far incomplete (1). Insights about the folding state and packing of biomolecules inside LDCs will aid in understanding the relevance of metastable liquid-liquid phase separation, as well as understanding initial processes of protein aggregation, oligomerization and misfolding, e.g. in neurodegenerative diseases like Alzheimer, Parkinson, dementia and sclerosis [2]. In order to investigate formation and internal structure of liquid dense clusters (LDC) by coherent X-ray diffractive imaging, selected distinct proteins of high medical and biological relevance as well as RNAs were selected to prepare LDCs. The physico-chemical conditions for the formation, stabilization and labelling of selected macromolecule LDCs were established and systematically optimized. The influence of polyvalent ions and polyethylene glycols as precipitant agents, which can promote LDC formation, were investigated. A combination of single particle Brownian microscopy applying an ultrafast camera, in situ Dynamic Light Scattering as well as fluorescens and atomic force microscopy have been applied to follow the time resolved process of clusters formation. Initial results and data obtained were utilized to understand and to optimize the clusters formation, as well as assess their stability, also towards advanced X-ray imaging techniques. More detailed information about folding state and arrangement of macromolecules inside LDCs, particularly by using X-ray free electron laser based imaging will open new routes to utilize LDCs for structural investigation of nanomaterials and single macromolecules in a non-crystalline environment in the future, and will allow to prepare distinct nano- and micro-sized crystals, most suitable for serial diffraction data collection. Details will be presented.
References:

[1] Falke, S. et al. (2018). Enc Anal Chem. Accepted.

[2] B. Maziuk. et al. (2017). Front Mol Neurosci.10, 89.

Keywords: liquid dense clusters, crystal nucleation