Journal: Proc Natl Acad Sci U S A / Year: 2015 Title: Structural basis of human γ-secretase assembly. Authors: Linfeng Sun / Lingyun Zhao / Guanghui Yang / Chuangye Yan / Rui Zhou / Xiaoyuan Zhou / Tian Xie / Yanyu Zhao / Shenjie Wu / Xueming Li / Yigong Shi / Abstract: The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ- ...The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.
History
Deposition
Apr 3, 2015
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Header (metadata) release
May 6, 2015
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Map release
Jun 17, 2015
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Update
Aug 12, 2015
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Current status
Aug 12, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Organism: Homo sapiens (human) / Recombinant cell: HEK 293S
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
4.2 mg/mL
Buffer
pH: 7.4 Details: 0.1% digitonin, 25 mM HEPES, pH 7.4, and 150 mM NaCl.
Grid
Details: Quantifoil Cu R1.2/1.3 grids
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging
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Electron microscopy
Microscope
FEI TITAN KRIOS
Date
Dec 22, 2014
Image recording
Category: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Number real images: 3312 / Average electron dose: 4.5 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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