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

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Basic information

Entry
Database: PDB / ID: 7ad9
TitleStructure of the Lifeact-F-actin complex
Components
  • Actin, alpha skeletal muscle
  • Lifeact
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton / Actin / Lifeact / labelling.
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 / filamentous actin ...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 / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle 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 / lamellipodium / protein-macromolecule adaptor activity / cell body / hydrolase activity / viral translational frameshifting / 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
Phalloidin / ADENOSINE-5'-DIPHOSPHATE / PHOSPHATE ION / Actin, alpha skeletal muscle / tRNA(Thr) (cytosine(32)-N(3))-methyltransferase
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Saccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical 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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 28, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Lifeact
B: Actin, alpha skeletal muscle
C: Lifeact
D: Actin, alpha skeletal muscle
L: Lifeact
H: Actin, alpha skeletal muscle
E: Lifeact
F: Actin, alpha skeletal muscle
G: Lifeact
I: Actin, alpha skeletal muscle
O: Phalloidin
P: Phalloidin
Q: Phalloidin
R: Phalloidin
S: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)226,96330
Polymers224,23115
Non-polymers2,73215
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27480 Å2
ΔGint-290 kcal/mol
Surface area72750 Å2
MethodPISA

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Components

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Protein/peptide , 2 types, 10 molecules ACLEGOPQRS

#1: Protein/peptide
Lifeact


Mass: 1927.243 Da / Num. of mol.: 5 / Source method: obtained synthetically
Details: Lifeact. Actin-binding peptide derived from the yeast protein ABP140
Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08641*PLUS
#3: Protein/peptide
Phalloidin


Type: Cyclic peptide / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: Phalloidin

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Protein , 1 types, 5 molecules BDHFI

#2: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 42109.973 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Details: Alpha-actin from rabbit skeletal muscle / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135

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Non-polymers , 3 types, 15 molecules

#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#6: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO4

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex between skeletal Actin from rabbit, Phalloidin and LifeActCOMPLEX#1-#30MULTIPLE SOURCES
2LifeActCOMPLEX#11RECOMBINANT
3ActinCOMPLEX#21NATURAL
4PhalloidinCOMPLEX#31RECOMBINANT
Molecular weightValue: 163 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Oryctolagus cuniculus (rabbit)9986
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
24synthetic construct (others)32630
Buffer solutionpH: 8
Details: 120 mM KCl, 20 mM Tris pH 8, 2 mM MgCl2, 1 mM DTT, and 0.02% w/v Tween-20
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMPotassion ChlorideKCl1
22 mMMagnesium ChlorideMgCl21
320 mMTris1
41 mMDTT1
50.02 % w/vTween-201
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 60 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 915

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9SPHIREinitial Euler assignment
10SPHIREfinal Euler assignment
12SPHIRE3D reconstruction
13Rosettamodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -167.18 ° / Axial rise/subunit: 27.3 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 246423
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 223480 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingPDB-ID: 6FHL

6fhl


Pdb chain-ID: C

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