Abstract
The ice nucleation protein InaZ of Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. We determine ab initio structures of this repeat segment, resolved to 1.1Å by microfocus x-ray crystallography and 0.9Å by the cryoEM method MicroED, from both racemic and homochiral crystals. We evaluate the benefits of racemic protein crystals for structure determination by MicroED and confirm that phase restriction introduced by crystal centrosymmetry increases the number of successful trials during ab initio phasing of electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel beta 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. Our determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with sub-micron sized crystals.
Synopsis The atomic asymmetry, left or right handedness, present in macromolecules and first described by Pasteur in his experiments with tartaric acid, is evident even in complex molecular assemblies like amyloid fibrils. Here, using the cryoEM method MicroED, we show that a segment from the ice nucleation protein InaZ assembles into homochiral and racemic water-binding amyloid protofibrils.
Footnotes
Funding information U.S. Department of Energy (grant No. DE-FC02-02ER63421); National Institutes of Health, National Institute of General Medical Sciences (grant No. P41 GM103403 and No. R35 GM128867); National Science Foundation, Office of Integrative Activities (grant No. DMR 1548924); Arnold and Mabel Beckman Foundation (award to Jose A. Rodriguez); Searle Scholars Program (award to Jose A. Rodriguez); Pew Charitable Trusts (award to Jose A. Rodriguez); QCB Collaboratory (award to Marcus Gallagher-Jones); National Institutes of Health, Division of Biomedical Research Workforce (award No. GM007185 to Calina Glynn); Howard Hughes Medical Institute (award to Tamir Gonen).