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- EMDB-7831: Cryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized... -

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Basic information

Entry
Database: EMDB / ID: EMD-7831
TitleCryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized F-actin
Map dataSymmetrized map of FLNaABD-E254K bound to phalloidin-stabilized F-actin. This map has been low-pass filtered to 3.6 A and sharpened with a B-factor of -150.
Sample
  • Complex: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin
    • Protein or peptide: Filamin-A
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin-binding domain / Actin crosslinker / STRUCTURAL PROTEIN
Function / homology
Function and homology information


regulation of membrane repolarization during atrial cardiac muscle cell action potential / regulation of membrane repolarization during cardiac muscle cell action potential / establishment of Sertoli cell barrier / Myb complex / glycoprotein Ib-IX-V complex / adenylate cyclase-inhibiting dopamine receptor signaling pathway / formation of radial glial scaffolds / positive regulation of integrin-mediated signaling pathway / actin crosslink formation / tubulin deacetylation ...regulation of membrane repolarization during atrial cardiac muscle cell action potential / regulation of membrane repolarization during cardiac muscle cell action potential / establishment of Sertoli cell barrier / Myb complex / glycoprotein Ib-IX-V complex / adenylate cyclase-inhibiting dopamine receptor signaling pathway / formation of radial glial scaffolds / positive regulation of integrin-mediated signaling pathway / actin crosslink formation / tubulin deacetylation / blood coagulation, intrinsic pathway / protein localization to bicellular tight junction / OAS antiviral response / Striated Muscle Contraction / positive regulation of actin filament bundle assembly / positive regulation of neuron migration / Cell-extracellular matrix interactions / early endosome to late endosome transport / Fc-gamma receptor I complex binding / positive regulation of potassium ion transmembrane transport / apical dendrite / cell-cell junction organization / positive regulation of neural precursor cell proliferation / positive regulation of platelet activation / protein localization to cell surface / wound healing, spreading of cells / podosome / negative regulation of transcription by RNA polymerase I / megakaryocyte development / GP1b-IX-V activation signalling / receptor clustering / positive regulation of axon regeneration / cortical cytoskeleton / SMAD binding / RHO GTPases activate PAKs / actin filament bundle / brush border / semaphorin-plexin signaling pathway / striated muscle thin filament / skeletal muscle thin filament assembly / cilium assembly / blood vessel remodeling / mitotic spindle assembly / epithelial to mesenchymal transition / potassium channel regulator activity / axonal growth cone / heart morphogenesis / stress fiber / skeletal muscle fiber development / release of sequestered calcium ion into cytosol / positive regulation of substrate adhesion-dependent cell spreading / protein sequestering activity / regulation of cell migration / dendritic shaft / protein kinase C binding / protein localization to plasma membrane / actin filament / negative regulation of DNA-binding transcription factor activity / trans-Golgi network / mRNA transcription by RNA polymerase II / G protein-coupled receptor binding / establishment of protein localization / synapse organization / negative regulation of protein catabolic process / cerebral cortex development / small GTPase binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Z disc / kinase binding / platelet aggregation / positive regulation of protein import into nucleus / actin filament binding / cell-cell junction / Platelet degranulation / actin cytoskeleton / negative regulation of neuron projection development / actin cytoskeleton organization / toxin activity / GTPase binding / angiogenesis / DNA-binding transcription factor binding / perikaryon / transmembrane transporter binding / positive regulation of canonical NF-kappaB signal transduction / hydrolase activity / postsynapse / protein stabilization / cadherin binding / focal adhesion / negative regulation of apoptotic process / nucleolus / perinuclear region of cytoplasm / glutamatergic synapse / protein homodimerization activity / RNA binding / extracellular exosome / extracellular region / ATP binding / nucleus / membrane
Similarity search - Function
Amanitin/phalloidin toxin / Filamin family / Filamin/ABP280 repeat / Filamin-type immunoglobulin domains / Filamin/ABP280 repeat / Filamin/ABP280 repeat profile. / Filamin/ABP280 repeat-like / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site ...Amanitin/phalloidin toxin / Filamin family / Filamin/ABP280 repeat / Filamin-type immunoglobulin domains / Filamin/ABP280 repeat / Filamin/ABP280 repeat profile. / Filamin/ABP280 repeat-like / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Immunoglobulin E-set / Immunoglobulin-like fold
Similarity search - Domain/homology
Phalloidin proprotein / Filamin-A / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human) / Gallus gallus (chicken) / Amanita phalloides (death cap)
Methodhelical reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsIwamoto DV / Huehn AR
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) releaseAug 8, 2018-
Map releaseSep 19, 2018-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
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  • Surface view with fitted model
  • Atomic models: PDB-6d8c
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6d8c
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7831.png

Downloads & links

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Map

FileDownload / File: emd_7831.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetrized map of FLNaABD-E254K bound to phalloidin-stabilized F-actin. This map has been low-pass filtered to 3.6 A and sharpened with a B-factor of -150.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 210 pix.
= 279.3 Å		1.33 Å/pix.
x 210 pix.
= 279.3 Å		1.33 Å/pix.
x 210 pix.
= 279.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.17799109 - 0.40055856
Average (Standard dev.)0.0012726671 (±0.014981323)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 279.30002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z279.300279.300279.300
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.1780.4010.001

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Supplemental data

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Mask #1

Fileemd_7831_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Symmetrized map of FLNaABD-E254K bound to phalloidin-stabilized F-actin....

Fileemd_7831_additional.map
AnnotationSymmetrized map of FLNaABD-E254K bound to phalloidin-stabilized F-actin. Low-pass filtered to 10 A and sharpened with a B-factor of -150. Masked to display the FLNaABD CH2 domain cryo-EM density.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Independently refined half map(2) of FLNaABD-E254K bound to...

Fileemd_7831_half_map_1.map
AnnotationIndependently refined half map(2) of FLNaABD-E254K bound to phalloidin-stabilized F-actin. This half map was symmetrized using parameters calculated from the full 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) of FLNaABD-E254K bound to...

Fileemd_7831_half_map_2.map
AnnotationIndependently refined half map(1) of FLNaABD-E254K bound to phalloidin-stabilized F-actin. This half map was symmetrized using parameters calculated from the full map.
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 E254K bound to phalloidin-stabilized F...

EntireName: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin
Components
  • Complex: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin
    • Protein or peptide: Filamin-A
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F...

SupramoleculeName: Helical complex of FLNaABD E254K bound to phalloidin-stabilized F-actin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Filamin-A

MacromoleculeName: Filamin-A / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.47625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHGSL VPRSENLYFQ GSDILQGTMS SSHSRAGQSA AGAAPGGGVD TRDAEMPATE KDLAEDAPWK KIQQNTFTRW CNEHLKCVS KRIANLQTDL SDGLRLIALL EVLSQKKMHR KHNQRPTFRQ MQLENVSVAL EFLDRESIKL VSIDSKAIVD G NLKLILGL ...String:
MHHHHHHGSL VPRSENLYFQ GSDILQGTMS SSHSRAGQSA AGAAPGGGVD TRDAEMPATE KDLAEDAPWK KIQQNTFTRW CNEHLKCVS KRIANLQTDL SDGLRLIALL EVLSQKKMHR KHNQRPTFRQ MQLENVSVAL EFLDRESIKL VSIDSKAIVD G NLKLILGL IWTLILHYSI SMPMWDEEED EEAKKQTPKQ RLLGWIQNKL PQLPITNFSR DWQSGRALGA LVDSCAPGLC PD WDSWDAS KPVTNAREAM QQADDWLGIP QVITPEEIVD PNVDKHSVMT YLSQFPKAKL KPGAPLRPK

UniProtKB: Filamin-A

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Breast
Molecular weightTheoretical: 41.862613 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(HYP)AW(G5G)A(ALO)C

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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 TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 2140 / Average exposure time: 12.0 sec. / Average electron dose: 50.0 e/Å2
Details: Micrographs from only one of the grids were used in the reconstruction. From that grid, only micrographs where Gctf detected signal at resolutions better than 4A were used in the reconstruction (~15%).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 37500 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.54 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.73 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0.3) / Number images used: 67000
Segment selectionNumber selected: 67000 / Software - Name: EMAN2
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6c1d

chain_id: B, residue_range: 1-375, source_name: PDB, initial_model_type: experimental model

3hoc

chain_id: A, residue_range: 39-153, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6d8c:
Cryo-EM structure of FLNaABD E254K bound to phalloidin-stabilized F-actin

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