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- PDB-4ckg: Helical reconstruction of ACAP1(BAR-PH domain) decorated membrane... -

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Entry
Database: PDB / ID: 4ckg
TitleHelical reconstruction of ACAP1(BAR-PH domain) decorated membrane tubules by cryo-electron microscopy
ComponentsARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
KeywordsSIGNALING PROTEIN / MEMBRANE REMODELING
Function / homologyBAR domain of APPL family / ARFGAP/RecO-like zinc finger / Ankyrin repeat region circular profile. / ARF GTPase-activating proteins domain profile. / Ankyrin repeat profile. / PH domain profile. / Ankyrin repeats (3 copies) / Putative GTPase activating protein for Arf / PH domain / ArfGAP domain superfamily ...BAR domain of APPL family / ARFGAP/RecO-like zinc finger / Ankyrin repeat region circular profile. / ARF GTPase-activating proteins domain profile. / Ankyrin repeat profile. / PH domain profile. / Ankyrin repeats (3 copies) / Putative GTPase activating protein for Arf / PH domain / ArfGAP domain superfamily / Ankyrin repeat-containing domain superfamily / AH/BAR domain superfamily / Ankyrin repeat-containing domain / PH-like domain superfamily / Ankyrin repeat / Pleckstrin homology domain / Arf GTPase activating protein / GTPase activator activity / 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
Specimen sourceHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 15 Å resolution
AuthorsPang, X.Y. / Fan, J. / Zhang, Y. / Zhang, K. / Gao, B.Q. / Ma, J. / Li, J. / Deng, Y.C. / Zhou, Q.J. / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Jan 6, 2014 / Release: Oct 15, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 15, 2014Structure modelrepositoryInitial release
1.1Oct 22, 2014Structure modelDatabase references
1.2Aug 30, 2017Structure modelData collection / Derived calculations / Refinement descriptionem_3d_fitting / em_software / pdbx_struct_assembly / pdbx_struct_assembly_gen_em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _pdbx_struct_assembly.details / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.oper_expression
1.3Jan 23, 2019Structure modelAdvisory / Data collection / Derived calculationspdbx_validate_close_contact / struct_conn / struct_conn_type

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2546
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Assembly

Deposited unit
A: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
B: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
C: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
D: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1


Theoretical massNumber of molelcules
Total (without water)173,3374
Polyers173,3374
Non-polymers00
Water0
1
A: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
B: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
C: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
D: ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1
x 10


Theoretical massNumber of molelcules
Total (without water)1,733,37440
Polyers1,733,37440
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation9

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Components

#1: Protein/peptide
ARF-GAP WITH COILED-COIL, ANK REPEAT AND PH DOMAIN-CONTAINING PROTEIN 1 / CENTAURIN-BETA-1 / CNT-B1 / BAR-PH DOMAIN OF ACAP1


Mass: 43334.348 Da / Num. of mol.: 4 / Fragment: BAR-PH DOMAIN, RESIDUES 1-377 / Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid name: PGEX-6P-1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q15027
Sequence detailsPFAM PF03114

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / Reconstruction method: helical reconstruction

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Sample preparation

ComponentName: BAR-PH DOMAIN OF ACAP1 / Type: COMPLEX / Details: MICROGRAPHS SELECTED MANUALLY
Buffer solutionName: 50MM HEPES, PH7.4, 100MM NACL / Details: 50MM HEPES, PH7.4, 100MM NACL / pH: 7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS / Date: Jul 16, 2012
Details: GOOD MICROGRAPHS (VERIFIED BY HELICAL DIFFRACTION PATTERN) WERE SELECTED MANUALLY. THE ELECTRON DOSE IS 2000 ELECTRON PER NANOMETER SQUARE.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 / Calibrated magnification: 125418 / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm
Specimen holderTemperature: 98 kelvins
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNumber digital images: 259
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategoryDetails
1UCSF Chimeramodel fittingChimera plus manual docking
2IHRSR3D reconstruction
CTF correctionDetails: CTFFIND3
3D reconstructionMethod: HELICAL RECONSTRUCTION USING IHRSR WITH THE PARTICLES SHRUNK 4 TIMES TO IMPROVE THE ACCURACY OF ALIGNMENT.
Resolution: 15 Å / Number of particles: 367 / Nominal pixel size: 3.6 / Actual pixel size: 3.6
Details: PARTICLES WERE CLASSIFIED BY THEIR DIAMETERS, THEN SHRUNK 4 TIMES TO PERFORM SINGLE PARTICLE RECONSTRUCTION USING IHRSR. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2546. (DEPOSITION ID: 12221).
Symmetry type: HELICAL
Atomic model buildingDetails: METHOD--RIGID BODY FITTING AND MANUALLY DOCKING REFINEMENT PROTOCOL--X-RAY
Ref protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 4NSW
Least-squares processHighest resolution: 12 Å
Refine hist #LASTHighest resolution: 12 Å
Number of atoms included #LASTProtein: 11682 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 11682

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