National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
United States
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)
United States
Citation
Journal: Sci Adv / Year: 2021 Title: Structural insights into integrin αβ opening by fibronectin ligand. Authors: Stephanie Schumacher / Dirk Dedden / Roberto Vazquez Nunez / Kyoko Matoba / Junichi Takagi / Christian Biertümpfel / Naoko Mizuno / Abstract: Integrin αβ is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and αβ undergo conformational changes. While this is key for cell-tissue connections, ...Integrin αβ is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and αβ undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human αβ with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The αβ-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting αβ adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of αβ for fibronectin is increased with manganese ions (Mn) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and αβ opening is induced by ligand-binding.
History
Deposition
Mar 18, 2021
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Header (metadata) release
Aug 25, 2021
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Map release
Aug 25, 2021
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Update
Aug 25, 2021
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Current status
Aug 25, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 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
0.15 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
50.0 mM
Tris
tris(hydroxymethyl)aminomethane
150.0 mM
NaCl
sodium chloride
1.0 mM
MgCl2
manganese chloride
1.0 mM
CaCl2
calcium chloride
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: GloQube 20 mA
Vitrification
Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details
Integrin a5b1 was assembled into MSPE3D1 nanodiscs that contained bovine brain lipids (Folch fraction I) at a ratio of 1:29:3460, respectively. The assembly mix was incubated with SM-2 BioBeads to remove DDM detergent from solubilized lipids and then purified by size-exclusion chromatography using a Superose 6 3.2/300 column.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 1829 / #0 - Average exposure time: 7.39 sec. / #0 - Average electron dose: 65.8 e/Å2 #0 - Details: movie: 20 frames/image dose per frame: 3.13 e-/A2 #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 989 / #1 - Average exposure time: 2.5 sec. / #1 - Average electron dose: 82.3 e/Å2 #1 - Details: movie: 50 frames/image dose per frame: 1.65 e-/A2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
initial image processing automatically by FOCUS pipeline: gain normalization, motion correction and dose-weighting in MotionCor2 CTF estimation by GCTF
Particle selection
Number selected: 496356 Details: 1424 particles were manually picked and used to obtain 2D classes with distinct features as templates for Gautomatch
CTF correction
Software - Name: Gctf
Startup model
Type of model: OTHER Details: 30 A-low pass filtered model from previous data analysis: 502 particles were manually picked from 2600 micrographs and used for 2D classification to generate a template for gautomatch, which ...Details: 30 A-low pass filtered model from previous data analysis: 502 particles were manually picked from 2600 micrographs and used for 2D classification to generate a template for gautomatch, which picked 103534 particles. After 2D classification an initial model was obtained in Cryosparc. Further 3D classification and refinement in Relion used 23332 particles yielded a 3D map that was used as a starting model for the high-resolution data set.
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 98750
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classification
Number classes: 8 / Avg.num./class: 30000 / Software - Name: RELION (ver. 3.0) / Details: 255514 particle after initial sorting
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