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- EMDB-8161: Nervous wreck (Nwk aa 1-731) on lipid monolayer -

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Basic information

Entry
Database: EMDB / ID: EMD-8161
TitleNervous wreck (Nwk aa 1-731) on lipid monolayer
Map dataNervous wreck (Nwk aa 1-731) on lipid monolayer
Sample
  • Cell: Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal tag, on membrane monolayer
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsRodal AA / Sokolova OS / Stanishneva-Konovalova TB
Funding support United States, Russian Federation, 3 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1420382 United States
RSF14-14-00234 Russian Federation
Pew Charitable Trustsnone United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.
Authors: Tatiana B Stanishneva-Konovalova / Charlotte F Kelley / Tania L Eskin / Emily M Messelaar / Steven A Wasserman / Olga S Sokolova / Avital A Rodal /
Abstract: Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis ...Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.
History
DepositionApr 22, 2016-
Header (metadata) releaseJul 13, 2016-
Map releaseSep 7, 2016-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8161.png

Downloads & links

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Map

FileDownload / File: emd_8161.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNervous wreck (Nwk aa 1-731) on lipid monolayer
Voxel sizeX=Y=Z: 3.4 Å
Density
Contour LevelBy EMDB: 0.44 / Movie #1: 0.45
Minimum - Maximum-0.114943534 - 0.6
Average (Standard dev.)0.0076197926 (±0.050582502)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 435.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.43.43.4
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z435.200435.200435.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.1150.6000.008

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Supplemental data

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Sample components

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Entire : Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal...

EntireName: Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal tag, on membrane monolayer
Components
  • Cell: Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal tag, on membrane monolayer

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Supramolecule #1: Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal...

SupramoleculeName: Nervous wreck (Nwk aa 1-731), containing a 6-hisXpress N-terminal tag, on membrane monolayer
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.06 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES, 100 mM NaCl
StainingType: NEGATIVE / Material: 1% uranyl acetate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 9.0 e/Å2

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Image processing

CTF correctionSoftware - Name: EMAN (ver. 2.1)
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Tilt angle: 45 degrees
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: OTHER / Software - Name: FREALIGN (ver. 9.09)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: FREALIGN (ver. 9.09) / Number images used: 4050

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