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- EMDB-2237: Electron cyro-microscopy helical reconstruction of Par-3 N-termin... -

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Basic information

Entry
Database: EMDB / ID: EMD-2237
TitleElectron cyro-microscopy helical reconstruction of Par-3 N-terminal domain
Map dataReconstruction of Par3NTD
Sample
  • Sample: Par-3 N-terminal DUF3534 domain
  • Protein or peptide: Par-3 N-terminal DUF3534 domain
Keywordscell polarity / DUF3534 domain / self-association
Function / homology
Function and homology information


Tight junction interactions / regulation of actin filament-based process / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / internode region of axon / PAR polarity complex / regulation of cellular localization / apical constriction / establishment of centrosome localization / lateral loop / positive regulation of myelination ...Tight junction interactions / regulation of actin filament-based process / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / internode region of axon / PAR polarity complex / regulation of cellular localization / apical constriction / establishment of centrosome localization / lateral loop / positive regulation of myelination / establishment of epithelial cell polarity / Schmidt-Lanterman incisure / bicellular tight junction assembly / myelination in peripheral nervous system / phosphatidylinositol-3-phosphate binding / establishment or maintenance of epithelial cell apical/basal polarity / protein targeting to membrane / wound healing, spreading of cells / centrosome localization / apical junction complex / establishment of cell polarity / negative regulation of peptidyl-threonine phosphorylation / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of receptor internalization / bicellular tight junction / endomembrane system / axonal growth cone / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / adherens junction / protein localization / microtubule cytoskeleton organization / spindle / cell-cell junction / apical part of cell / cell junction / cell cortex / protein phosphatase binding / cell adhesion / cell cycle / apical plasma membrane / cell division / neuronal cell body / protein-containing complex / identical protein binding / cytoplasm
Similarity search - Function
Par3/HAL, N-terminal / N-terminal of Par3 and HAL proteins / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily
Similarity search - Domain/homology
Partitioning defective 3 homolog
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsYan Z / Wenjuan W / Jia C / Kai Z / Feng G / Weimin G / Mingjie Z / Fei S / Wei F
CitationJournal: Structure / Year: 2013
Title: Structural insights into the intrinsic self-assembly of Par-3 N-terminal domain.
Authors: Yan Zhang / Wenjuan Wang / Jia Chen / Kai Zhang / Feng Gao / Bingquan Gao / Shuai Zhang / Mingdong Dong / Flemming Besenbacher / Weimin Gong / Mingjie Zhang / Fei Sun / Wei Feng /
Abstract: Par-3, the central organizer of the Par-3/Par-6/atypical protein kinase C complex, is a multimodular scaffold protein that is essential for cell polarity establishment and maintenance. The N-terminal ...Par-3, the central organizer of the Par-3/Par-6/atypical protein kinase C complex, is a multimodular scaffold protein that is essential for cell polarity establishment and maintenance. The N-terminal domain (NTD) of Par-3 is capable of self-association to form filament-like structures, although the underlying mechanism is poorly understood. Here, we determined the crystal structure of Par-3 NTD and solved the filament structure by cryoelectron microscopy. We found that an intrinsic "front-to-back" interaction mode is important for Par-3 NTD self-association and that both the lateral and longitudinal packing within the filament are mediated by electrostatic interactions. Disruptions of the lateral or longitudinal packing significantly impaired Par-3 NTD self-association and thereby impacted the Par-3-mediated epithelial polarization. We finally demonstrated that a Par-3 NTD-like domain from histidine ammonia-lyase also harbors a similar self-association capacity. This work unequivocally provides the structural basis for Par-3 NTD self-association and characterizes one type of protein domain that can self-assemble via electrostatic interactions.
History
DepositionDec 1, 2012-
Header (metadata) releaseJan 9, 2013-
Map releaseOct 16, 2013-
UpdateOct 16, 2013-
Current statusOct 16, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zee
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2237.tif

Downloads & links

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Map

FileDownload / File: emd_2237.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Par3NTD
Voxel sizeX=Y=Z: 0.933 Å
Density
Contour LevelBy AUTHOR: 5.2 / Movie #1: 5.2
Minimum - Maximum-12.060205460000001 - 18.52397728
Average (Standard dev.)0.0 (±1.26795149)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-180-180-180
Dimensions360360360
Spacing360360360
CellA=B=C: 335.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9330.9330.933
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z335.880335.880335.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-180-180-180
NC/NR/NS360360360
D min/max/mean-12.06018.524-0.000

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Supplemental data

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Sample components

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Entire : Par-3 N-terminal DUF3534 domain

EntireName: Par-3 N-terminal DUF3534 domain
Components
  • Sample: Par-3 N-terminal DUF3534 domain
  • Protein or peptide: Par-3 N-terminal DUF3534 domain

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Supramolecule #1000: Par-3 N-terminal DUF3534 domain

SupramoleculeName: Par-3 N-terminal DUF3534 domain / type: sample / ID: 1000 / Oligomeric state: helical filament assembly / Number unique components: 95
Molecular weightTheoretical: 938 KDa

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Macromolecule #1: Par-3 N-terminal DUF3534 domain

MacromoleculeName: Par-3 N-terminal DUF3534 domain / type: protein_or_peptide / ID: 1 / Name.synonym: Par-3 NTD / Number of copies: 95 / Oligomeric state: helical filament assembly / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway Rat
Molecular weightTheoretical: 938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET32a

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 8 / Details: 50 mM Tris, 100 mM NaCl, 1 mM DTT and 1 mM EDTA
GridDetails: 300-mesh GiGTM holy carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: blotted 3.0 s with a blot force 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 85 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 96,000 times magnification
Legacy - Electron beam tilt params: 0
DateDec 1, 2010
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 6460 / Average electron dose: 20 e/Å2
Details: The images were automatically collected by using leginon system.
Bits/pixel: 32
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 96000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe initial model was obtained IHRSR. Then particles were aligned using EMAN1.
Final reconstructionApplied symmetry - Helical parameters - Δz: 3.53 Å
Applied symmetry - Helical parameters - Δ&Phi: 43.84 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: OTHER / Software - Name: Spider, EMAN1
Details: The resolution was assessed by splitting original particles set into two halves and comparing two independent reconstructions. The final map was reconstructed by using the whole set.
CTF correctionDetails: each image
Final angle assignmentDetails: Euler angle system in EMAN1

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Atomic model buiding 1

Initial modelPDB ID:

4i6p


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera and NAMD2
DetailsProtocol: Rigid body fitting first and then molecular dynamics flexible fitting. Crystal structure of the monomer was docked as a rigid body into the cryoEM map using UCSF Chimera and applied with the helical symmetry to build the initial model. Then the structural model was solvated in a box of water molecules with 150 mM NaCl in VMD, using 15 angstrom of padding in all directions. Extra ions were added to neutralize the systems. The simulations were performed with program NAMD 2.8, using the CHARMM27 force field with CMAP corrections. All the fitting procedure is the same as the application of symmetry-restrained MDFF to chaperonin reported previously.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross correlation coefficient
Output model

PDB-3zee:
Electron cyro-microscopy helical reconstruction of Par-3 N terminal domain

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