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- EMDB-7833: FLNaABD-WT bound to phalloidin-stabilized F-actin -

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Basic information

Entry
Database: EMDB / ID: EMD-7833
TitleFLNaABD-WT bound to phalloidin-stabilized F-actin
Map dataMap of FLNaABD-wt bound to phalloidin-stabilized F-actin. Particles were selected for occupancy and the final map was not symmetrized. Low-pass filtered to 10A with a B-factor of -400.
Sample
  • Complex: Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin
    • Protein or peptide: Filamin A Actin-Binding Domain
    • Protein or peptide: Actin
Biological speciesHomo sapiens (human) / Gallus gallus (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsIwamoto DV / Huehn AR / Simon B / Huet-Calderwood C / Baldassarre M / Sindelar CV / Calderwood DA
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Structural basis of the filamin A actin-binding domain interaction with F-actin.
Authors: Daniel V Iwamoto / Andrew Huehn / Bertrand Simon / Clotilde Huet-Calderwood / Massimiliano Baldassarre / Charles V Sindelar / David A Calderwood /
Abstract: Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology ...Actin-cross-linking proteins assemble actin filaments into higher-order structures essential for orchestrating cell shape, adhesion, and motility. Missense mutations in the tandem calponin homology domains of their actin-binding domains (ABDs) underlie numerous genetic diseases, but a molecular understanding of these pathologies is hampered by the lack of high-resolution structures of any actin-cross-linking protein bound to F-actin. Here, taking advantage of a high-affinity, disease-associated mutant of the human filamin A (FLNa) ABD, we combine cryo-electron microscopy and functional studies to reveal at near-atomic resolution how the first calponin homology domain (CH1) and residues immediately N-terminal to it engage actin. We further show that reorientation of CH2 relative to CH1 is required to avoid clashes with actin and to expose F-actin-binding residues on CH1. Our data explain localization of disease-associated loss-of-function mutations to FLNaCH1 and gain-of-function mutations to the regulatory FLNaCH2. Sequence conservation argues that this provides a general model for ABD-F-actin binding.
History
DepositionApr 26, 2018-
Header (metadata) releaseJul 25, 2018-
Map releaseSep 19, 2018-
UpdateOct 17, 2018-
Current statusOct 17, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7833.png

Downloads & links

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Map

FileDownload / File: emd_7833.map.gz / Format: CCP4 / Size: 226.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of FLNaABD-wt bound to phalloidin-stabilized F-actin. Particles were selected for occupancy and the final map was not symmetrized. Low-pass filtered to 10A with a B-factor of -400.
Voxel sizeX=Y=Z: 1.247 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.009371536 - 0.05993219
Average (Standard dev.)0.0005838682 (±0.004139976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions390390390
Spacing390390390
CellA=B=C: 486.33 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2471.2471.247
M x/y/z390390390
origin x/y/z0.0000.0000.000
length x/y/z486.330486.330486.330
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS390390390
D min/max/mean-0.0090.0600.001

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Supplemental data

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Mask #1

Fileemd_7833_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Independently refined half map(1)

Fileemd_7833_half_map_1.map
AnnotationIndependently refined half map(1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Independently refined half map(2)

Fileemd_7833_half_map_2.map
AnnotationIndependently refined half map(2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin

EntireName: Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin
Components
  • Complex: Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin
    • Protein or peptide: Filamin A Actin-Binding Domain
    • Protein or peptide: Actin

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Supramolecule #1: Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin

SupramoleculeName: Helical complex of FLNaABD-WT bound to phalloidin-stabilized F-actin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Filamin A Actin-Binding Domain

MacromoleculeName: Filamin A Actin-Binding Domain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PATEKDLAED APWKKIQQNT FTRWCNEHLK CVSKRIANLQ TDLSDGLRLI ALLEVLSQKK MHRKHNQRPT FRQMQLENVS VALEFLDRE SIKLVSIDSK AIVDGNLKLI LGLIWTLILH YS

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Macromolecule #2: Actin

MacromoleculeName: Actin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Breast
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIEHG IITNWDDMEK IWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVTHNV P IYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIEHG IITNWDDMEK IWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVTHNV P IYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSYEL PD GQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITALA PST MKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKC

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 28.1 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.75 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Real space helical symmetry was not imposed on the final map
Number images used: 24000
FSC plot (resolution estimation)

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