Macromolecular structural dynamics visualized by pulsed dose control in 4D electron microscopy

Abstract

Macromolecular conformation dynamics, which span a wide range of time scales, are fundamental to the understanding of properties and functions of their structures. Here, we report direct imaging of structural dynamics of helical macromolecules over the time scales of conformational dynamics (ns to subsecond) by means of four-dimensional (4D) electron microscopy in the single-pulse and stroboscopic modes. With temporally controlled electron dosage, both diffraction and real-space images are obtained without irreversible radiation damage. In this way, the order-disorder transition is revealed for the organic chain polymer. Through a series of equilibrium-temperature and temperature-jump dependencies, it is shown that the metastable structures and entropy of conformations can be mapped in the nonequilibrium region of a "funnellike" free-energy landscape. The T -jump is introduced through a substrate (a "hot plate" type arrangement) because only the substrate is made to absorb the pulsed energy. These results illustrate the promise of ultrafast 4D imaging for other applications in the study of polymer physics as well as in the visualization of biological phenomena.materials | polymers | biopolymers M acromolecular structural dynamics, unlike those of small molecular systems, involve complex free-energy landscapes with numerous possible conformations (1, 2). A prime example is that of protein folding that occurs as a result of a search from a high-entropy state of many conformations to the low-entropy native structure (3-5). This process is best described by the balance between the entropic and enthalpic free-energy contributions, as well as the "diffusion" through a multitude of energy barriers that form nucleation centers or misfolded structures on the path to the final state. The behavior has been likened to phase transitions, and in this regard it is also applicable to other large systems including organic polymers (6-8).In the transition, these macromolecules maintain the constituents or sequence of repeating units, but the conformations must undergo rearrangements from, for example, folded chains to random coils or extended chains (9). It is, therefore, important to understand conformational dynamics of macromolecules, biological (10) or abiological (11), and to elucidate the nature of the free-energy landscape that exhibits the multiple metastable states that are separated by entropic and enthalpic barriers. Visualization of the structures can be achieved using both real-space and diffraction imaging of electron microscopy, but these macromolecules are organics with facile bonds and, thus, probing of their motions represents a real challenge because of radiation damage. Moreover, the probing must span the time scales involved, from ps for rotational orientation to ns and microsecond (μs) for barrier crossings, and to hundreds of ms for refolding into the native structure.Here, we report real-time visualization of helical-macromolecular dynamics using diffraction and real-space imaging methodology of four-dimen...

Dominance of misfolded intermediates in the dynamics of α-helix folding

“…Unlike the normal (Arrheniustype) behavior, in which the rates typically increase as the temperature increases, for macromolecules with many intermediates on the energy landscape raising the initial temperature (and measuring the rate) for a helical polymer structure or the α-helix refolding results in a slowdown of the relaxation process and hence the decrease in the rates (33,34).…”

Enhanced Optoelectronic Performance of a Passivated Nanowire‐Based Device: Key Information from Real‐Space Imaging Using 4D Electron Microscopy

“…A major advancement in improving the spatial resolution of time‐resolved techniques was achieved with the development of four dimensional (4D) ultrafast transmission electron microscopy as a new method for materials visualization. This technique is capable of an unprecedented spatial resolution that cannot be achieved by any purely optical techniques . Very recently, as a new direction in 4D electron microscopy, a second‐generation scanning ultrafast electron microscope (S‐UEM) with temporal resolution of 650 fs was developed, providing the unique opportunity to selectively visualize the dynamics of charge carriers at the material surface, which is inaccessible by both transmission electron microscopy and ultrafast laser spectroscopy .…”

Chirped imaging pulses in four-dimensional electron microscopy: femtosecond pulsed hole burning

“…The advent of four-dimensional (4D) electron microscopy has made possible the direct study of structural dynamics with atomic-scale spatiotemporal resolutions [1,2]. The scope of applications is diverse, including the study of chemical bonding dynamics [3], macromolecular conformation changes [4] and nanomechanical vibrations [5]; for a review see [6] and references therein. In these studies, ultrashort electron pulses are utilized in imaging, diffraction and spectroscopy, and it is essential to optimize the spatial and temporal coherence in order to achieve atomic-scale resolutions.…”