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- EMDB-3192: Helical reconstruction of amphiphysin N-BAR with a membrane tube ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3192
TitleHelical reconstruction of amphiphysin N-BAR with a membrane tube radius of 140 Angstrom by cryo-electron microscopy
Map dataReconstruction of amphiphysin N-BAR with a radius of 140 Angstrom
Sample
  • Sample: Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom
  • Protein or peptide: Amphiphysin
Keywordsamphiphysin / N-BAR / amphiphysin/BIN1 / BIN1
Function / homology
Function and homology information


rhabdomere membrane biogenesis / rhabdomere development / Clathrin-mediated endocytosis / Neutrophil degranulation / intracellular organelle / regulation of muscle contraction / exocytosis / cleavage furrow / phospholipid binding / protein localization ...rhabdomere membrane biogenesis / rhabdomere development / Clathrin-mediated endocytosis / Neutrophil degranulation / intracellular organelle / regulation of muscle contraction / exocytosis / cleavage furrow / phospholipid binding / protein localization / synapse / plasma membrane / cytoplasm
Similarity search - Function
Amphiphysin / Amphiphysin / BAR domain / BAR domain profile. / BAR / BAR domain / AH/BAR domain superfamily / SH3 domain / Src homology 3 domains / SH3-like domain superfamily ...Amphiphysin / Amphiphysin / BAR domain / BAR domain profile. / BAR / BAR domain / AH/BAR domain superfamily / SH3 domain / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain
Similarity search - Domain/homology
Biological speciesDrosophila melanogaster (fruit fly)
Methodhelical reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsAdam J / Basnet N / Mizuno N
CitationJournal: Sci Rep / Year: 2015
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
DepositionOct 5, 2015-
Header (metadata) releaseOct 28, 2015-
Map releaseOct 28, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0404
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0404
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0404
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emdb3192_500...

Downloads & links

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Map

FileDownload / File: emd_3192.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of amphiphysin N-BAR with a radius of 140 Angstrom
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.82 Å/pix.
x 300 pix.
= 546. Å		1.82 Å/pix.
x 300 pix.
= 546. Å		1.82 Å/pix.
x 300 pix.
= 546. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0404 / Movie #1: 0.0404
Minimum - Maximum-0.27337459 - 0.76345432
Average (Standard dev.)0.0 (±0.070712)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 546.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.821.821.82
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z546.000546.000546.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2730.7630.000

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Supplemental data

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Sample components

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Entire : Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom

EntireName: Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom
Components
  • Sample: Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom
  • Protein or peptide: Amphiphysin

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Supramolecule #1000: Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom

SupramoleculeName: Amphiphysin N-BAR with a membrane tube radius of 140 Angstrom
type: sample / ID: 1000 / Oligomeric state: polymer / Number unique components: 1

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Macromolecule #1: Amphiphysin

MacromoleculeName: Amphiphysin / type: protein_or_peptide / ID: 1 / Name.synonym: Amph / Oligomeric state: Homodimer / Recombinant expression: Yes
Source (natural)Organism: Drosophila melanogaster (fruit fly) / synonym: Fruit Fly / Location in cell: T-Tubules
Molecular weightExperimental: 60 KDa / Theoretical: 60 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: Gold / Recombinant plasmid: pEC
SequenceUniProtKB: Amphiphysin / InterPro: Amphiphysin

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.4 / Details: 20 mM Hepes, 500 mM NaCl, 1 mM EDTA, 1 mM DTT
GridDetails: Quantifoil R 2/1, Cu, 300 mesh grids
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Method: 10 s of manual blotting before plunging
Details20 uM of Amph N-BAR protein was incubated with 720 uM liposomes at RT.

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Name: GIF 200
DateAug 19, 2014
Image recordingCategory: 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsHelix 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 reconstructionApplied symmetry - Helical parameters - Δz: 3.81 Å
Applied symmetry - Helical parameters - Δ&Phi: 55.92 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.3 Å / Resolution method: OTHER / Software - Name: BSOFT, EMAN2, Relion, SPIDER, IHRSR
Details: For the reconstruction 1948 segmented particles were used. The particles were 2D classified by Relion and for the reconstruction the helical symmetry was applied using IHRSR.
CTF correctionDetails: Phases of individual images are flipped

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Atomic model buiding 1

Initial modelPDB ID:

1uru

SoftwareName: Chimera
DetailsThe amphiphysin N-BAR dimer was fitted by manual docking using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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