Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface.

Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 Å resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD

Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction.

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

Developments of electron diffraction phasing methods and their applications

Crystallization and phase retrieval are the two perennial problems that challenge any endeavor to retrieve structural information from a crystal. Micro-electron diffraction has emerged in recent years as a complementary method to X-ray diffraction by allowing the diffraction of crystals previously too small for routine X-ray techniques. While micro-electron diffraction has great potential to address barriers in crystallization, issues with phase retrieval precludes the technique’s broad utilization. As a nascent technique, many X-ray phasing methods have not been replicated in electron diffraction. In this work, we demonstrate the development and application of racemic crystallography and fragment-based phasing methods for electron diffraction

Developments of electron diffraction phasing methods and their applications

Crystallization and phase retrieval are the two perennial problems that challenge any endeavor to retrieve structural information from a crystal. Micro-electron diffraction has emerged in recent years as a complementary method to X-ray diffraction by allowing the diffraction of crystals previously too small for routine X-ray techniques. While micro-electron diffraction has great potential to address barriers in crystallization, issues with phase retrieval precludes the technique’s broad utilization. As a nascent technique, many X-ray phasing methods have not been replicated in electron diffraction. In this work, we demonstrate the development and application of racemic crystallography and fragment-based phasing methods for electron diffraction

Ab initio determination of peptide structures by MicroED

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 micro-diffraction (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 amyloid-forming 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

Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data

Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ

The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Å by microfocus X-ray crystallography and to 0.9 Å by the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel β-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals

Fragment-Based Ab Initio Phasing of Peptidic Nanocrystals by MicroED

Electron diffraction (MicroED/3DED) can render the three-dimensional atomic structures of molecules from previously unamenable samples. The approach has been particularly transformative for peptidic structures, where MicroED has revealed novel structures of naturally occurring peptides, synthetic protein fragments, and peptide-based natural products. Despite its transformative potential, MicroED is beholden to the crystallographic phase problem, which challenges its de novo determination of structures. ARCIMBOLDO, an automated, fragment-based approach to structure determination, eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptide structures including fragments of human amyloids, and yeast and mammalian prions. For electron diffraction, fragment-based phasing portends a more general phasing solution with limited model bias for a wider set of chemical structures.This work was performed as part of STROBE, an NSF Science and Technology Center through Grant DMR-1548924. This work is also supported by DOE Grant DE-FC02-02ER63421 and NIH-NIGMS Grant R35 GM128867 and P41GM136508. L.S.R. is supported by the USPHS National Research Service Award 5T32GM008496. M.D.F. was funded by Eugene V. Cota-Robles Fellowship and Ruth L. Kirschstein NRSA GM007185 and is currently funded by a Whitcome Pre-Doctoral Fellowship and a National Science Foundation Graduate Research Fellowship. C.G. was funded by Ruth L. Kirschstein NRSA GM007185 and is currently funded by Ruth L. Kirschstein Predoctoral Individual NRSA, 1F31 AI143368. R.J.B. received fellowship from FAPESP (16/24191-8 and 17/13485-3). C.M. is grateful to MICINN for her BES-2015-071397 scholarship associated with the Structural Biology Maria de Maeztu Unit of Excellence. This work was supported by grants PGC2018-101370-B-I00 and PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) and Generalitat de Catalunya (2017SGR-1192) to I.U. J.A.R. is supported as a Pew Scholar, a Beckman Young Investigator, and a Packard Fellow. The Northeastern Collaborative Access Team beamline is funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357