Chapter 17
Ab Initio Determination of Peptide Structures by MicroED
Chih-Te Zee, Ambarneil Saha, Michael R. Sawaya, and Jose A. Rodriguez
Abstract
Structural elucidation of small macromolecules such as peptides has recently been facilitated by a growing
number of technological advances to existing crystallographic methods. The emergence of electron microdiffraction (MicroED) of protein nanocrystals under cryogenic conditions has enabled the interrogation of
crystalline peptide assemblies only hundreds of nanometers thick. Collection of atomic or near-atomic
resolution data by these methods has permitted the ab initio determination of structures of various amyloidforming peptides, including segments derived from prions and ice-nucleating proteins. This chapter focuses
on the process of ab initio structural determination from nano-scale peptide assemblies and other similar
molecules.
Key words Peptide, Nanocrystal, Amyloid, Ab initio, Direct methods
1 Introduction
Electron micro-diffraction (MicroED) is a cryoEM technique wellsuited to the determination of peptide and small macromolecule
structures [1–4]. The first novel macromolecular structure solved
by MicroED [5], a peptide segment of the Parkinson’s-associated
protein alpha synuclein, established the method’s utility as a means
of rapid structural analysis of crystals as small as a hundred nanometers thick. The subsequent determination of four ab initio
structures of prion peptides demonstrated the approach’s expediency in obtaining atomic-resolution structures of peptides [6].
Since these initial breakthroughs, the investigation of peptides
by MicroED has grown and now includes over 20 structures;
14 structures at better than 1.2 A ˚ resolution [3, 7–11]. Because
of the success of ab initio methods for structural determination of
peptide assemblies by MicroED [3, 6], we focus this chapter on
procedures for the collection of high-resolution MicroED data
from peptide nanocrystals and atomic-resolution determination of
ab initio structures from said crystals. This approach hinges on the
availability of well-ordered crystals hundreds of nanometers thick as
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
329
Ab Initio Determination of Peptide Structures by MicroED
Chih-Te Zee, Ambarneil Saha, Michael R. Sawaya, and Jose A. Rodriguez
Abstract
Structural elucidation of small macromolecules such as peptides has recently been facilitated by a growing
number of technological advances to existing crystallographic methods. The emergence of electron microdiffraction (MicroED) of protein nanocrystals under cryogenic conditions has enabled the interrogation of
crystalline peptide assemblies only hundreds of nanometers thick. Collection of atomic or near-atomic
resolution data by these methods has permitted the ab initio determination of structures of various amyloidforming peptides, including segments derived from prions and ice-nucleating proteins. This chapter focuses
on the process of ab initio structural determination from nano-scale peptide assemblies and other similar
molecules.
Key words Peptide, Nanocrystal, Amyloid, Ab initio, Direct methods
1 Introduction
Electron micro-diffraction (MicroED) is a cryoEM technique wellsuited to the determination of peptide and small macromolecule
structures [1–4]. The first novel macromolecular structure solved
by MicroED [5], a peptide segment of the Parkinson’s-associated
protein alpha synuclein, established the method’s utility as a means
of rapid structural analysis of crystals as small as a hundred nanometers thick. The subsequent determination of four ab initio
structures of prion peptides demonstrated the approach’s expediency in obtaining atomic-resolution structures of peptides [6].
Since these initial breakthroughs, the investigation of peptides
by MicroED has grown and now includes over 20 structures;
14 structures at better than 1.2 A ˚ resolution [3, 7–11]. Because
of the success of ab initio methods for structural determination of
peptide assemblies by MicroED [3, 6], we focus this chapter on
procedures for the collection of high-resolution MicroED data
from peptide nanocrystals and atomic-resolution determination of
ab initio structures from said crystals. This approach hinges on the
availability of well-ordered crystals hundreds of nanometers thick as
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
329