Journal: FEBS Lett / Year: 2016 Title: Nucleotide-dependent conformational changes of the AAA+ ATPase p97 revisited. Authors: Jan M Schuller / Florian Beck / Philip Lössl / Albert J R Heck / Friedrich Förster / Abstract: The ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, ...The ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-γS at 6.1-7.4 Å resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.
History
Deposition
Feb 3, 2016
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Header (metadata) release
Mar 9, 2016
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Map release
Mar 9, 2016
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Update
Mar 9, 2016
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Current status
Mar 9, 2016
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.4 / Details: Buffer: 40 mM Tris, 150 mM NaCl, 1 mM MgCl2
Grid
Details: Lacey Carbon grids
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 2 seconds before plunging
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Electron microscopy
Microscope
FEI TITAN KRIOS
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected at 105,000 times magnification
Specialist optics
Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Date
Nov 3, 2014
Image recording
Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1728 / Average electron dose: 40 e/Å2 Details: Every image is the average of 30 frames recorded by the direct electron detector
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
The particles were selected using an automatic selection program implemented in the TOM toolbox.
CTF correction
Details: On the micrograph level
Final reconstruction
Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: OTHER / Software - Name: RELION / Details: Map was corrected using a B-factor of -576 A^2. / Number images used: 40000
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