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- EMDB-11721: Structure of the Lifeact-F-actin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11721
TitleStructure of the Lifeact-F-actin complex
Map data
Sample
  • Complex: Complex between skeletal Actin from rabbit, Phalloidin and LifeAct
    • Complex: LifeActLifeAct Dye
      • Protein or peptide: LifeactLifeAct Dye
    • Complex: Actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate
Function / homology
Function and homology information


tRNAThr (cytosine32-N3)-methyltransferase / tRNA (cytidine-3-)-methyltransferase activity / tRNA methylation / mating projection tip / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle ...tRNAThr (cytosine32-N3)-methyltransferase / tRNA (cytidine-3-)-methyltransferase activity / tRNA methylation / mating projection tip / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / actin filament binding / protein-macromolecule adaptor activity / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
tRNA N(3)-methylcytidine methyltransferase METTL2/6/8-like / Methyltransferase type 12 / Methyltransferase domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family ...tRNA N(3)-methylcytidine methyltransferase METTL2/6/8-like / Methyltransferase type 12 / Methyltransferase domain / 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 / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Actin, alpha skeletal muscle / tRNA(Thr) (cytosine(32)-N(3))-methyltransferase
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Oryctolagus cuniculus (rabbit) / synthetic construct (others) / Rabbit (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBelyy A / Merino F / Sitsel O / Raunser S
CitationJournal: PLoS Biol / Year: 2020
Title: Structure of the Lifeact-F-actin complex.
Authors: Alexander Belyy / Felipe Merino / Oleg Sitsel / Stefan Raunser /
Abstract: Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been ...Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.
History
DepositionSep 14, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ad9
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ad9
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11721.png

Downloads & links

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Map

FileDownload / File: emd_11721.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.21 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.15678103 - 0.28605673
Average (Standard dev.)0.0015207349 (±0.009130586)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 363.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.211.211.21
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z363.000363.000363.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1570.2860.002

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Supplemental data

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Mask #1

Fileemd_11721_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map filtered according to local resolution

Fileemd_11721_additional_1.map
AnnotationMap filtered according to local resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11721_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11721_half_map_2.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 : Complex between skeletal Actin from rabbit, Phalloidin and LifeAct

EntireName: Complex between skeletal Actin from rabbit, Phalloidin and LifeAct
Components
  • Complex: Complex between skeletal Actin from rabbit, Phalloidin and LifeAct
    • Complex: LifeActLifeAct Dye
      • Protein or peptide: LifeactLifeAct Dye
    • Complex: Actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Phalloidin
      • Protein or peptide: Phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHATE IONPhosphate

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Supramolecule #1: Complex between skeletal Actin from rabbit, Phalloidin and LifeAct

SupramoleculeName: Complex between skeletal Actin from rabbit, Phalloidin and LifeAct
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 163 kDa/nm

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Supramolecule #2: LifeAct

SupramoleculeName: LifeAct / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #3: Actin

SupramoleculeName: Actin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #4: Phalloidin

SupramoleculeName: Phalloidin / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Lifeact

MacromoleculeName: Lifeact / type: protein_or_peptide / ID: 1
Details: Lifeact. Actin-binding peptide derived from the yeast protein ABP140
Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 1.927243 KDa
SequenceString:
MGVADLIKKF ESISKEE

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Details: Alpha-actin from rabbit skeletal muscle / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
Molecular weightTheoretical: 42.109973 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIE(HIC)G IIT NWDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLD SG DGVTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSS S LEKSYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQ KEITALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #3: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 808.899 Da
SequenceString:
(HYP)AW(EEP)A(DTH)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 / Adenosine diphosphate

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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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Macromolecule #6: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION / Phosphate

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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
Component:
ConcentrationFormulaName
100.0 mMKClPotassion Chloride
2.0 mMMgCl2Magnesium Chloride
20.0 mMTris
1.0 mMDTT
0.02 % w/vTween-20

Details: 120 mM KCl, 20 mM Tris pH 8, 2 mM MgCl2, 1 mM DTT, and 0.02% w/v Tween-20
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 915 / Average exposure time: 3.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 246423
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6fhl

Final angle assignmentType: NOT APPLICABLE / Software - Name: SPHIRE
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 27.3 Å
Applied symmetry - Helical parameters - Δ&Phi: -167.18 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 223480

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Atomic model buiding 1

Initial modelPDB ID:

6fhl


Chain - Chain ID: C
Output model

PDB-7ad9:
Structure of the Lifeact-F-actin complex

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