Journal: Elife / Year: 2020 Title: Structure and activation mechanism of the BBSome membrane protein trafficking complex. Authors: Sandeep K Singh / Miao Gui / Fujiet Koh / Matthew Cj Yip / Alan Brown / Abstract: Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS ...Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the β-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.
History
Deposition
Dec 19, 2019
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Header (metadata) release
Jan 29, 2020
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Map release
Jan 29, 2020
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Update
Mar 6, 2024
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Current status
Mar 6, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: CALCIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: CA
Molecular weight
Theoretical: 40.078 Da
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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
0.7 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Name
Formula
20.0 mM
Hepes
220.0 mM
NaCl
5.0 mM
MgCl2
4.0 mM
beta-mercaptoethanol
Details: The buffer also contained 36 uM ARL6 and 1 mM GTP.
Grid
Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR Details: The grids were glow discharged at 15 mA for 30 s with a PELCO easiGlow Glow Discharge Cleaning System.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK II / Details: Grids were blotted for 2 s with a -2 offset..
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 25 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 9408 / Average exposure time: 4.0 sec. / Average electron dose: 56.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type of model: OTHER Details: An initial map for the BBSome was generated using RELION's implementation of the stochastic gradient descent algorithm using default parameters and a mask diameter of 280 Angstrom.
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 152942
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classification
Number classes: 8 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)
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Atomic model buiding 1
Details
During refinement, the resolution limit was set to 3.1 Angstrom. Secondary structure, Ramachandran and rotamer restraints were applied during refinement.
Refinement
Space: REAL / Protocol: OTHER / Overall B value: 43.6 / Target criteria: Correlation coefficient
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