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Assessing structure and dynamics of biological systems via Liquid Phase Electron Microscopy 

Assessing structure and dynamics of biological systems via Liquid Phase Electron Microscopy 

Session

Stream 2: EMAG - In-situ microscopy

Authors

Dr Lorena Ruiz-Perez (1), Mr Cesare De Pace (1), Dr Silvia Acosta-Gutiérrez (1), Mr Gabriel Ing (1), Mr Gabriele Marchelo (1, 4), Dr Simona Pilotto (2), Prof Finn Werner (2), Prof Francesco L Gervasio (1, 5), Prof Giuseppe Battaglia (1, 3, 6)

Affiliations

1. University College London, Department of Chemistry
2. University College London, Department of Structural and Molecular Biology
3. Institute for Bioengineering of Catalunya (IBEC), The Barcelona Institute of Science and Technology
4. Italian Institute of Technology
5. University of Geneva, Pharmaceutical Sciences
6. Catalan Institution for Research and Advanced Studies (ICREA)

Keywords

low dose imaging, protein reconstruction, Liquid Phase TEM, in-situ TEM, protein dynamics, dynamics TEM

Abstract text

Recent developments in electron-transparent materials have paved the way for liquid-phase electron microscopy (LP-EM) and sample imaging in a liquid environment.  LP-EM  offers tremendous potential in a myriad of fields ranging from soft matter, nano-materials, polymer assemblies, biomaterials, synthetic biology, and design and catalysis to name a few. The liquid nature of the specimen presents exciting new prospects. It is well known that dispersed particles in a liquid undergo Brownian motion involving continuous translational displacement and rotation of the particles. Such rotation means that each particle dispersed in the liquid will show several profiles under the TEM disclosing potentially all of its surface. This phenomena can be exploited for structure reconstruction. 

Image reconstruction in liquid-state poses several challenges, and most importantly, it undermines the single-particle analysis assumption that the three-dimensional objects captured on the image sensor are identical. We propose the combination of all-atom simulations with LP-EM to complement structural studies with dynamic investigations. In this work, we exploited LP-EM to image the dynamics of particles undergoing Brownian motion, using their natural rotation to access the particle 3D conformational landscape. In this fashion 3D reconstructions of particles in liquid are performed  v using tomographic techniques. We have selected two case studies  for our approach according to prior data accessibility and physiological environmental factors: apoferritin and archaeal RNA polymerase. We show that our approach allows us to get sub-nanometer spatial resolutions of protein structures, either imaging one by one and assessing different conformational states or combining several proteins into one statistical conformational ensemble.

The work presented was accomplished using a Jeol JEM 2200FS equipped with an in-column omega filter in combination with the high performance in-situ camera from Gatan, the K2-IS. The ultra-high sensitivity of the K2 allows low-dose imaging modes limiting considerably the electron dose damage. The in-situ TEM holders used were the Ocean and Stream holders from DENSsolutions. 

References