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- EMDB-2546: Helical reconstruction of ACAP1(BAR-PH domain) decorated membrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-2546
TitleHelical reconstruction of ACAP1(BAR-PH domain) decorated membrane tubules by cryo-electron microscopy
Map dataReconstruction of the first class of BarPH with diameter of 43.2nm
Sample
  • Sample: BARPH domain of ACAP1
  • Protein or peptide: BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain
KeywordsACAP1 / BAR-PH domain / Electron microscopy / Membrane remodeling.
Function / homology
Function and homology information


GTPase activator activity / recycling endosome membrane / protein transport / membrane / metal ion binding
Similarity search - Function
ArfGAP ACAP1/2/3-like / BAR domain of APPL family / Arf GTPase activating protein / ArfGAP domain superfamily / Putative GTPase activating protein for Arf / ARF GTPase-activating proteins domain profile. / Putative GTP-ase activating proteins for the small GTPase, ARF / BAR domain / ARFGAP/RecO-like zinc finger / AH/BAR domain superfamily ...ArfGAP ACAP1/2/3-like / BAR domain of APPL family / Arf GTPase activating protein / ArfGAP domain superfamily / Putative GTPase activating protein for Arf / ARF GTPase-activating proteins domain profile. / Putative GTP-ase activating proteins for the small GTPase, ARF / BAR domain / ARFGAP/RecO-like zinc finger / AH/BAR domain superfamily / AH/BAR domain superfamily / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / Pleckstrin homology domain / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / PH-like domain superfamily
Similarity search - Domain/homology
Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsPang XY / Fan J / Zhang Y / Zhang K / Gao BQ / Ma J / Li J / Deng YC / Zhou QJ / Hsu V / Sun F
CitationJournal: Dev Cell / Year: 2014
Title: A PH domain in ACAP1 possesses key features of the BAR domain in promoting membrane curvature.
Authors: Xiaoyun Pang / Jun Fan / Yan Zhang / Kai Zhang / Bingquan Gao / Jun Ma / Jian Li / Yuchen Deng / Qiangjun Zhou / Edward H Egelman / Victor W Hsu / Fei Sun /
Abstract: The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane ...The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending.
History
DepositionJan 1, 2014-
Header (metadata) releaseJan 15, 2014-
Map releaseOct 15, 2014-
UpdateApr 18, 2018-
Current statusApr 18, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4ckg
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5h3d
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4ckg
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5h3d
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2546-cls...

Downloads & links

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Map

FileDownload / File: emd_2546.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the first class of BarPH with diameter of 43.2nm
Voxel sizeX=Y=Z: 3.6 Å
Density
Contour LevelBy AUTHOR: 0.00157 / Movie #1: 0.002
Minimum - Maximum-0.07147177 - 0.070062
Average (Standard dev.)0.00068746 (±0.01151327)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 720.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z720.000720.000720.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.0710.0700.001

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Supplemental data

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Sample components

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Entire : BARPH domain of ACAP1

EntireName: BARPH domain of ACAP1
Components
  • Sample: BARPH domain of ACAP1
  • Protein or peptide: BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain

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Supramolecule #1000: BARPH domain of ACAP1

SupramoleculeName: BARPH domain of ACAP1 / type: sample / ID: 1000
Details: 4 mg/ml ACAP1(BAR-PH) protein was incubated with 2 mg/ml liposome of 200nm at room temperature for 60min.
Oligomeric state: tetramer / Number unique components: 1
Molecular weightExperimental: 15.98 MDa / Theoretical: 15.98 MDa / Method: Theoretical computation

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Macromolecule #1: BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain

MacromoleculeName: BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain
type: protein_or_peptide / ID: 1 / Name.synonym: BAR-PH domain of ACAP1 / Number of copies: 107 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: endosome / Location in cell: Endosomal membrane
Molecular weightExperimental: 9.2 MDa / Theoretical: 9.2 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: PGEX-6P-1
SequenceUniProtKB: Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1
GO: recycling endosome membrane
InterPro: AH/BAR domain superfamily, Pleckstrin homology domain

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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

Concentration4 mg/mL
BufferpH: 7.4 / Details: 50mM HEPES, pH7.4, 100mM NaCl
GridDetails: 300-mesh GiG holy carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 98 K / Instrument: FEI VITROBOT MARK IV
Method: The grid was blotted 3.0 s with a blot force 2 before plunging.
Details4 mg/ml BARPH protein was incubated with 2mg/ml liposome of 200nm at room temperature for 60min.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 125418 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder: Liquid nitrogen / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 93 K / Max: 103 K / Average: 98 K
DateJul 16, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 259 / Average electron dose: 20 e/Å2 / Bits/pixel: 32
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3
Final angle assignmentDetails: The Euler angles were determined by the projection angle.
Final reconstructionApplied symmetry - Helical parameters - Δz: 23.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 93 °
Applied symmetry - Helical parameters - Axial symmetry: C3 (3 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: IHRSR
Details: The particles were shrunk 4 times to improve the alignment accuracy. Final maps were calculated from the datasets generated by 6 filaments with diameter of 43.2nm.
DetailsThe particles were aligned using IHRSR

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Atomic model buiding 1

Initial modelPDB ID:

4nsw


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera plus manual docking
DetailsThe ACAP1 dimer was separately fitted by manual docking and optimized using Chimera. Other dimers were generated by applying helical symmetry to the fitted one.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-4ckg:
Helical reconstruction of ACAP1(BAR-PH domain) decorated membrane tubules by cryo-electron microscopy

PDB-5h3d:
Helical structure of membrane tubules decorated by ACAP1 (BARPH doamin) protein by cryo-electron microscopy and MD simulation

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