Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction

Abstract

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three−dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three−dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three−dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction.

Serial protein crystallography in an electron microscope

“…A further improvement would be achieved using specialized TEM grids 44 , or microfabricated chips 45 . The SerialED approach could also be applied to heterogeneous systems with extended amorphous regions, such as cells containing in vivo grown nanocrystals 3 , or to map and exploit local lattice structures 46 . Similarly, mixtures of crystals within a single grid or contaminated samples can be studied without significant modifications by assigning each found lattice to one of the contained sample classes using multiple indexing runs or direct classification of diffraction patterns 36,47 .…”

Serial protein crystallography in an electron microscope

“…A further mitigation might be achieved using specialized TEM grids 44 , or microfabricated chips 45 . The SerialED-approach could also be applied to heterogeneous systems with extended amorphous regions, such as cells containing in-vivo grown nanocrystals 3 , or to map and exploit local lattice structures 46 .…”

“…While this has not been performed in the present work, it may prove beneficial for cases where clear boundaries of adjacent crystals are not readily discernible, or to study and exploit local lattice structures 46,68 .…”

Data-driven challenges and opportunities in crystallography

“…These include ultra-bright ultra-fast X-ray sources, upgraded synchrotron facilities and accessible instruments for routine electron diffraction. Single experiments can now yield gigabytes or terabytes of data from one or more samples allowing unprecedented exploration of ultra-fast processes or from increasingly small crystallites [2][3][4]. Here, we discuss two areas of rapid growth in structural biology that are increasingly reliant on large-scale data gathering: serial X-ray crystallography [1,5,6] and electron crystallography of micro and nanocrystals [7][8][9][10].…”