[English] 日本語
![](img/lk-miru.gif)
- EMDB-3194: Helical reconstruction of amphiphysin N-BAR with a membrane tube ... -
+
Open data
-
Basic information
Entry | Database: EMDB / ID: EMD-3194 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Helical reconstruction of amphiphysin N-BAR with a membrane tube radius of 131 Angstrom by cryo-electron microscopy | |||||||||
![]() | Reconstruction of amphiphysin N-BAR with a radius of 131 Angstrom | |||||||||
![]() |
| |||||||||
![]() | amphiphysin / N-BAR / amphiphysin/BIN1 / BIN1 | |||||||||
Function / homology | ![]() rhabdomere membrane biogenesis / organelle / rhabdomere development / Clathrin-mediated endocytosis / Neutrophil degranulation / regulation of muscle contraction / exocytosis / cleavage furrow / phospholipid binding / protein localization ...rhabdomere membrane biogenesis / organelle / rhabdomere development / Clathrin-mediated endocytosis / Neutrophil degranulation / regulation of muscle contraction / exocytosis / cleavage furrow / phospholipid binding / protein localization / synapse / plasma membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 10.9 Å | |||||||||
![]() | Adam J / Basnet N / Mizuno N | |||||||||
![]() | ![]() Title: Structural insights into the cooperative remodeling of membranes by amphiphysin/BIN1. Authors: Julia Adam / Nirakar Basnet / Naoko Mizuno / ![]() Abstract: Amphiphysin2/BIN1 is a crescent-shaped N-BAR protein playing a key role in forming deeply invaginated tubes in muscle T-tubules. Amphiphysin2/BIN1 structurally stabilizes tubular formations in ...Amphiphysin2/BIN1 is a crescent-shaped N-BAR protein playing a key role in forming deeply invaginated tubes in muscle T-tubules. Amphiphysin2/BIN1 structurally stabilizes tubular formations in contrast to other N-BAR proteins involved in dynamic membrane scission processes; however, the molecular mechanism of the stabilizing effect is poorly understood. Using cryo-EM, we investigated the assembly of the amphiphysin/BIN1 on a membrane tube. We found that the N-BAR domains self-assemble on the membrane surface in a highly cooperative manner. Our biochemical assays and 3D reconstructions indicate that the N-terminal amphipathic helix H0 plays an important role in the initiation of the tube assembly and further in organizing BAR-mediated polymerization by locking adjacent N-BAR domains. Mutants that lack H0 or the tip portion, which is also involved in interactions of the neighboring BAR unit, lead to a disruption of the polymer organization, even though tubulation can still be observed. The regulatory region of amphiphysin/BIN1 including an SH3 domain does not have any apparent involvement in the polymer lattice. Our study indicates that the H0 helix and the BAR tip are necessary for efficient and organized self-assembly of amphiphysin/N-BAR. | |||||||||
History |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
-
Downloads & links
-EMDB archive
Map data | ![]() | 90.7 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 10.4 KB 10.4 KB | Display Display | ![]() |
Images | ![]() | 978.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
-
Links
EMDB pages | ![]() ![]() |
---|---|
Related items in Molecule of the Month |
-
Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of amphiphysin N-BAR with a radius of 131 Angstrom | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.82 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-
Sample components
-Entire : Amphiphysin N-BAR with a membrane tube radius of 131 Angstrom
Entire | Name: Amphiphysin N-BAR with a membrane tube radius of 131 Angstrom |
---|---|
Components |
|
-Supramolecule #1000: Amphiphysin N-BAR with a membrane tube radius of 131 Angstrom
Supramolecule | Name: Amphiphysin N-BAR with a membrane tube radius of 131 Angstrom type: sample / ID: 1000 / Oligomeric state: polymer / Number unique components: 1 |
---|
-Macromolecule #1: Amphiphysin N-BAR
Macromolecule | Name: Amphiphysin N-BAR / type: protein_or_peptide / ID: 1 / Name.synonym: Amph N-BAR / Oligomeric state: Dimer / Recombinant expression: Yes |
---|---|
Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Amphiphysin / InterPro: Amphiphysin |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
![]() | helical reconstruction |
Aggregation state | helical array |
-
Sample preparation
Concentration | 0.6 mg/mL |
---|---|
Buffer | pH: 7.4 / Details: 20 mM Hepes, 500 mM NaCl, 1 mM EDTA, 1 mM DTT |
Grid | Details: Quantifoil R 2/1, Cu, 300 mesh grids |
Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Method: 10 s of manual blotting before plunging |
Details | 20 uM of Amph N-BAR protein was incubated with 720 uM liposomes at RT. |
-
Electron microscopy
Microscope | FEI POLARA 300 |
---|---|
Specialist optics | Energy filter - Name: GIF 200 |
Date | Aug 19, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 271 / Average electron dose: 30 e/Å2 Details: every image is average of 30 frames recorded by the direct electron detector |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000 |
Sample stage | Specimen holder model: OTHER |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
-
Image processing
Details | Helix handedness is not confirmed by sub-tomogram averaging. Used programs: BSOFT software package, particle picking by EMAN2 with e2helixboxer, 2D classification by Relion, 3D helical reconstruction by IHRSR implemented into SPIDER. |
---|---|
Final reconstruction | Applied symmetry - Helical parameters - Δz: 3.84 Å Applied symmetry - Helical parameters - Δ&Phi: 56.01 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.9 Å / Resolution method: OTHER / Software - Name: BSOFT, EMAN2, Relion, SPIDER, IHRSR Details: For the reconstruction 692 segmented particles were used. The particles were 2D classified by Relion and for the reconstruction the helical symmetry was applied using IHRSR. |
CTF correction | Details: Phases of individual images are flipped |
-Atomic model buiding 1
Initial model | PDB ID: |
---|---|
Software | Name: ![]() |
Details | The amphiphysin N-BAR dimer was fitted by manual docking using Chimera. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |