|Entry||Database: EMDB / ID: 2546|
|Title||Helical reconstruction of ACAP1(BAR-PH domain) decorated membrane tubules by cryo-electron microscopy|
|Map data||Reconstruction of the first class of BarPH with diameter of 43.2nm|
|Sample||BARPH domain of ACAP1|
|Keywords||ACAP1 / BAR-PH domain / Electron microscopy / Membrane remodeling.|
|Function / homology||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 ...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
|Source||Homo sapiens (human)|
|Method||helical reconstruction / cryo EM / 15 Å resolution|
|Authors||Pang XY / Fan J / Zhang Y / Zhang K / Gao BQ / Ma J / Li J / Deng YC / Zhou QJ / Hsu V / Sun F|
|Citation||Journal: 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 Report||PDB-ID: 4ckg|
About validation report
|Date||Deposition: Jan 1, 2014 / Header (metadata) release: Jan 15, 2014 / Map release: Oct 15, 2014 / Last update: Apr 18, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_2546.map.gz (map file in CCP4 format, 31251 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.6 Å|
CCP4 map header:
-Entire BARPH domain of ACAP1
|Entire||Name: 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
|Mass||Theoretical: 15.98 MDa / Experimental: 15.98 MDa / Measured by: Theoretical computation|
-Component #1: protein, BAR-PH domain of ArfGAP with coiled coil, ANK repeat and...
|Protein||Name: 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
|Mass||Theoretical: 9.2 MDa / Experimental: 9.2 MDa|
|Source||Species: 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 references||InterPro: 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
|Specimen||Specimen state: helical array / Method: cryo EM|
|Helical parameters||Axial symmetry: C3 (3 fold cyclic) / Hand: LEFT HANDED / Delta z: 23.2 Å / Delta phi: 93 deg.|
|Crystal grow details||4 mg/ml BARPH protein was incubated with 2mg/ml liposome of 200nm at room temperature for 60min.|
|Sample solution||Specimen conc.: 4 mg/ml / Buffer solution: 50mM HEPES, pH7.4, 100mM NaCl / pH: 7.4|
|Support film||300-mesh GiG holy carbon grid|
|Vitrification||Instrument: 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.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Jul 16, 2012|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 75000 X (nominal), 125418 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2500 - 3500 nm|
|Specimen Holder||Holder: Liquid nitrogen / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 98 K ( 93 - 103 K)|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 259 / Sampling size: 15 microns / Bit depth: 32|
|Processing||Method: helical reconstruction / Details: The particles were aligned using IHRSR|
|3D reconstruction||Algorithm: 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
-Atomic model buiding
|Modeling #1||Software: 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
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