Advances in methods for atomic resolution macromolecular structure determination

Abstract

Recent technical advances have dramatically increased the power and scope of structural biology. New developments in high-resolution cryo-electron microscopy, serial X-ray crystallography, and electron diffraction have been especially transformative. Here we highlight some of the latest advances and current challenges at the frontiers of atomic resolution methods for elucidating the structures and dynamical properties of macromolecules and their complexes.

Keywords: Structural biology; computational methods; electron diffraction; electron microscopy; x-ray crystallography.

Figure 1.. Deposition of atomic or near-atomic-resolution structures in the Protein Data Bank according to different experimental methods.

( A) The number of structures deposited annually is shown between 2010 and the end of 2019, based on X-ray diffraction, cryo-electron microscopy (EM), and NMR methods. A sub-4 Å resolution criterion was imposed (for X-ray and EM). The inset shows the depositions since 2015 on a logarithmic scale. ( B) The same set of structures are broken down by resolution. Those based on EM do not include those determined by electron diffraction methods (see Figure 4). This figure is an original image created by the authors for this publication.

Figure 2.. High-resolution cryo-electron microscopy (EM) as a tool for drug discovery.

Cryo-EM density at a resolution of approximately 2.6 Å reveals a streptogramin antibiotic (green sticks) bound to the peptidyl transferase center (PTC) of a bacterial ribosome (wheat sticks), as described by Li, Pellegrino, et al. ( https://doi.org/10.26434/chemrxiv.8346107.v2). The map is shown as a blue volume and is rendered only within 2 Å of the antibiotic for clarity ( A). Views of the streptogramin molecule (ribosome deleted) normal to ( B) and coplanar with ( C) the macrocycle ring illustrate features such as the proline in the macrocycle backbone, isopropyl side chain, and carbonyl groups, allowing unambiguous placement of the drug in order to inform structure-based design. This figure is an original image created by the authors for this publication.

Figure 3.. Sample delivery strategies for serial crystallography.

In the microfluidic variety of the experiment ( A), crystals are delivered to the X-ray beam using a microfluidic nozzle ranging from tens to hundreds of microns in diameter. A stream of randomly oriented microcrystals continuously flows perpendicular to the pulsing X-ray beam (black arrow). In the fixed-target version ( B), microcrystals are mounted (by pipetting) in random orientations on a solid support chip that contains appropriately sized windows, and the chip is rapidly moved through the pulsing X-ray beam (black arrow) by automated translation in the x and y directions. In both diagrams, each crystal yields a single diffraction pattern capturing a random slice of reciprocal space. Crystals that have not been probed by the X-ray beam are colored green, and those that have been measured, and destroyed, are depicted in purple. In time-resolved serial crystallography, a perturbation (shown by the red arrow) is applied to the crystals with user-defined timing prior to the X-ray pulse ( Δt). This figure is an original image created by the authors for this publication.

Figure 4.. A survey of biomolecular structures determined by electron diffraction from three-dimensional crystals.

An aggregate analysis of Protein Data Bank (PDB) depositions shows the breakdown of novel versus known structures and the distribution of structures according to their reported resolution (in Å). Cartoon diagrams of several representative structures are shown; below each structure are properties (molecular weight and resolution) as well as identifiers (date associated with deposition release and PDB code). This figure is an original image created by the authors for this publication.