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

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Entry
Database: EMDB / ID: 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
SampleBARPH domain of ACAP1
  • BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain
KeywordsACAP1 / BAR-PH domain / Electron microscopy / Membrane remodeling.
Function / homologyAH/BAR domain superfamily / Ankyrin repeat / ArfGAP domain superfamily / ARFGAP/RecO-like zinc finger / Ankyrin repeat-containing domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat-containing domain / PH-like domain superfamily / Pleckstrin homology domain / Putative GTPase activating protein for Arf ...AH/BAR domain superfamily / Ankyrin repeat / ArfGAP domain superfamily / ARFGAP/RecO-like zinc finger / Ankyrin repeat-containing domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat-containing domain / PH-like domain superfamily / Pleckstrin homology domain / Putative GTPase activating protein for Arf / Arf GTPase activating protein / PH domain profile. / Ankyrin repeat profile. / ARF GTPase-activating proteins domain profile. / Ankyrin repeat region circular profile. / PH domain / AH/BAR domain superfamily / GTPase activator activity / Pleckstrin homology domain / protein transport / recycling endosome membrane / membrane / metal ion binding / Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1
Function and homology information
SourceHomo sapiens (human)
Methodhelical reconstruction / cryo EM / 15 Å resolution
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.
Validation ReportPDB-ID: 4ckg

SummaryFull report
PDB-ID: 5h3d

SummaryFull report
About validation report
DateDeposition: Jan 1, 2014 / Header (metadata) release: Jan 15, 2014 / Map release: Oct 15, 2014 / Last update: Apr 18, 2018

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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 models: PDB-4ckg
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-5h3d
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Movie viewer
Structure viewerEM map:
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Supplemental images

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Map

Fileemd_2546.map.gz (map file in CCP4 format, 31251 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
3.6 Å/pix.
= 720. Å
200 pix
3.6 Å/pix.
= 720. Å
200 pix
3.6 Å/pix.
= 720. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.6 Å
Density
Contour Level:0.00157 (by author), 0.002 (movie #1):
Minimum - Maximum-0.07147177 - 0.07006200
Average (Standard dev.)0.00068746 (0.01151327)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin-100-100-100
Limit999999
Spacing200200200
CellA=B=C: 720.0 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
Details: 4 mg/ml ACAP1(BAR-PH) protein was incubated with 2 mg/ml liposome of 200nm at room temperature for 60min.
Number of components: 1 / Oligomeric State: tetramer
MassTheoretical: 15.98 MDa / Experimental: 15.98 MDa / Measured by: Theoretical computation

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

ProteinName: BAR-PH domain of ArfGAP with coiled coil, ANK repeat and PH domain
a.k.a: BAR-PH domain of ACAP1 / Oligomeric Details: dimer / Recombinant expression: Yes / Number of Copies: 107
MassTheoretical: 9.2 MDa / Experimental: 9.2 MDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: PGEX-6P-1 / Strain: BL21(DE3)
Source (natural)Organelle: endosome / Location in cell: Endosomal membrane
External referencesInterPro: AH/BAR domain superfamily, Pleckstrin homology domain
UniProt: Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 1
Gene Ontology: recycling endosome membrane

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C3 (3 fold cyclic) / Hand: LEFT HANDED / Delta z: 23.2 Å / Delta phi: 93 deg.
Crystal grow details4 mg/ml BARPH protein was incubated with 2mg/ml liposome of 200nm at room temperature for 60min.
Sample solutionSpecimen conc.: 4 mg/ml / Buffer solution: 50mM HEPES, pH7.4, 100mM NaCl / pH: 7.4
Support film300-mesh GiG holy carbon grid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 98 K / Humidity: 100 %
Method: The grid was blotted 3.0 s with a blot force 2 before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jul 16, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000 X (nominal), 125418 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2500 - 3500 nm
Specimen HolderHolder: Liquid nitrogen / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 98 K ( 93 - 103 K)
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 259 / Sampling size: 15 microns / Bit depth: 32

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Image processing

ProcessingMethod: helical reconstruction / Details: The particles were aligned using IHRSR
3D reconstructionAlgorithm: Helical reconstruction by IHRSR
Euler angles: The Euler angles were determined by the projection angle.
Software: IHRSR / CTF correction: CTFFIND3
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.
Resolution: 15 Å / Resolution method: OTHER

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Atomic model buiding

Modeling #1Software: Chimera plus manual docking / Refinement protocol: rigid body / Target criteria: cross correlation / Refinement space: REAL
Details: The ACAP1 dimer was separately fitted by manual docking and optimized using Chimera. Other dimers were generated by applying helical symmetry to the fitted one.
Input PDB model: 4NSW
Chain ID: A, B
Output model

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