National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41 GM103403
United States
Citation
Journal: IUCrJ / Year: 2019 Title: Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ. Authors: Chih-Te Zee / Calina Glynn / Marcus Gallagher-Jones / Jennifer Miao / Carlos G Santiago / Duilio Cascio / Tamir Gonen / Michael R Sawaya / Jose A Rodriguez / Abstract: The ice-nucleation protein InaZ from contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. structures of this repeat segment, ...The ice-nucleation protein InaZ from contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. 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 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.
Details: Batch Mode: 0.1 M McIlvaine, pH 4.2, 12.5% w/v PEG8000, 0.1 M sodium chloride Temperature: 298 K
Buffer solution
pH: 4.2
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Cryogen name: ETHANE
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Data collection
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
Microscopy
Model: FEI TECNAI F20
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: DIFFRACTION
Image recording
Electron dose: 0.01 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffraction
Camera length: 1156 mm
EM diffraction shell
Resolution: 7.27→0.9 Å / Fourier space coverage: 86.4 % / Multiplicity: 4.3 / Num. of structure factors: 1747 / Phase residual: 1.0E-10 °
EM diffraction stats
Details: Phase statistics are not applicable. No imaging was used. The phases were obtained using a crystallographic direct methods program, SHELXD. Fourier space coverage: 86.4 % / High resolution: 0.9 Å / Num. of intensities measured: 7494 / Num. of structure factors: 1750 / Phase error: 0 ° / Phase residual: 1.0E-10 ° / Phase error rejection criteria: not applicable / Rmerge: 16.2 / Rsym: 16.2
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