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- EMDB-2034: Twofold symmetrized reconstruction of the FLNa16-21 rod2 segment ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2034
TitleTwofold symmetrized reconstruction of the FLNa16-21 rod2 segment bound with integrin Beta7 subunit tail
Map dataThis is a twofold symmetrized reconstruction of the filamin rod2 segment comprising domains 16-21 in the presence of a tenfold stoichiometric excess of integrin Beta7 subunit tail
Sample
  • Sample: Filamin A rod-2 segment, comprising domains 16-21 bound with an integrin Beta7 subunit tail
  • Protein or peptide: FLNa16-21-Beta7 tail
Keywordscell adhesion / integrin signaling / signal transduction / single particle
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.8 Å
AuthorsHofmann GW / Jiang P / Campbell ID / Gilbert RJC
CitationJournal: Biochem J / Year: 2012
Title: The C-terminal rod 2 fragment of filamin A forms a compact structure that can be extended.
Authors: Salla Ruskamo / Robert Gilbert / Gregor Hofmann / Pengju Jiang / Iain D Campbell / Jari Ylänne / Ulla Pentikäinen /
Abstract: Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several ...Filamins are large proteins that cross-link actin filaments and connect to other cellular components. The C-terminal rod 2 region of FLNa (filamin A) mediates dimerization and interacts with several transmembrane receptors and intracellular signalling adaptors. SAXS (small-angle X-ray scattering) experiments were used to make a model of a six immunoglobulin-like domain fragment of the FLNa rod 2 (domains 16-21). This fragment had a surprising three-branched structural arrangement, where each branch was made of a tightly packed two-domain pair. Peptides derived from transmembrane receptors and intracellular signalling proteins induced a more open structure of the six domain fragment. Mutagenesis studies suggested that these changes are caused by peptides binding to the CD faces on domains 19 and 21 which displace the preceding domain A-strands (18 and 20 respectively), thus opening the individual domain pairs. A single particle cryo-EM map of a nine domain rod 2 fragment (domains 16-24), showed a relatively compact dimeric particle and confirmed the three-branched arrangement as well as the peptide-induced conformation changes. These findings reveal features of filamin structure that are important for its interactions and mechanical properties.
History
DepositionJan 12, 2012-
Header (metadata) releaseSep 26, 2012-
Map releaseSep 26, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2034.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a twofold symmetrized reconstruction of the filamin rod2 segment comprising domains 16-21 in the presence of a tenfold stoichiometric excess of integrin Beta7 subunit tail
Voxel sizeX=Y=Z: 2.37 Å
Density
Contour LevelBy AUTHOR: 10.0 / Movie #1: 10
Minimum - Maximum-53.138774869999999 - 89.718048100000004
Average (Standard dev.)0.55061495 (±5.24991512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 236.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.372.372.37
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z237.000237.000237.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS100100100
D min/max/mean-53.13989.7180.551

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Supplemental data

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Sample components

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Entire : Filamin A rod-2 segment, comprising domains 16-21 bound with an i...

EntireName: Filamin A rod-2 segment, comprising domains 16-21 bound with an integrin Beta7 subunit tail
Components
  • Sample: Filamin A rod-2 segment, comprising domains 16-21 bound with an integrin Beta7 subunit tail
  • Protein or peptide: FLNa16-21-Beta7 tail

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Supramolecule #1000: Filamin A rod-2 segment, comprising domains 16-21 bound with an i...

SupramoleculeName: Filamin A rod-2 segment, comprising domains 16-21 bound with an integrin Beta7 subunit tail
type: sample / ID: 1000 / Oligomeric state: Dimer / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa

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Macromolecule #1: FLNa16-21-Beta7 tail

MacromoleculeName: FLNa16-21-Beta7 tail / type: protein_or_peptide / ID: 1 / Name.synonym: Activated filamin A rod 2
Details: The sample was imaged as a monodisperse distribution in vitreous ice
Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm (adjacent plasma membrane)
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
GridDetails: 300 mesh copper with lacey carbon
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home made guillotine
Method: Whatman No. 1 paper blot for 1-2 seconds prior to plunging.

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 110 K
Alignment procedureLegacy - Astigmatism: At 112,000 times magnification
DateSep 1, 2006
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 48 / Od range: 5 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final angle assignmentDetails: SPIDER Euler angles.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, SPIDER
Details: The fineness of sampling was restricted to 15 degrees to reflect inaccuracy of angle determination at this particle size at finer spacings. The resolution of the map was therefore fixed at 18 Angstrom.
Number images used: 32477
DetailsThe raw images were treated with a three-level decomposition with a fourth-order bi-orthogonal wavelet function in MATLAB to enhance contrast. Particles were selected semi-automatically using Boxer.

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