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- PDB-7bte: Lifeact-F-actin complex -

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Basic information

Entry
Database: PDB / ID: 7bte
TitleLifeact-F-actin complex
Components
  • Actin, alpha skeletal muscle
  • Lifeact
KeywordsCONTRACTILE PROTEIN/PROTEIN BINDING / F-actin / ADP-F-actin / CONTRACTILE PROTEIN-PROTEIN BINDING complex
Function / homology
Function and homology information


tRNAThr (cytosine32-N3)-methyltransferase / tRNA (cytidine-3-)-methyltransferase activity / Striated Muscle Contraction / tRNA methylation / mating projection tip / actin filament bundle / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber ...tRNAThr (cytosine32-N3)-methyltransferase / tRNA (cytidine-3-)-methyltransferase activity / Striated Muscle Contraction / tRNA methylation / mating projection tip / actin filament bundle / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin filament binding / actin cytoskeleton / protein-macromolecule adaptor activity / hydrolase activity / viral translational frameshifting / ATP 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
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle / tRNA(Thr) (cytosine(32)-N(3))-methyltransferase
Similarity search - Component
Biological speciesGallus gallus (chicken)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKumari, A. / Ragunath, V.K. / Sirajuddin, M.
Funding support India, 2items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/14/2/501533 India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: EMBO J / Year: 2020
Title: Structural insights into actin filament recognition by commonly used cellular actin markers.
Authors: Archana Kumari / Shubham Kesarwani / Manjunath G Javoor / Kutti R Vinothkumar / Minhajuddin Sirajuddin /
Abstract: Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely ...Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.
History
DepositionApr 1, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 29, 2020Group: Database references / Derived calculations / Category: citation / pdbx_struct_conn_angle / struct_conn
Item: _citation.journal_volume / _pdbx_struct_conn_angle.ptnr1_auth_asym_id ..._citation.journal_volume / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
G: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
L: Lifeact
M: Lifeact
N: Lifeact
hetero molecules


Theoretical massNumber of molelcules
Total (without water)218,52418
Polymers216,2668
Non-polymers2,25810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, TIRF
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18940 Å2
ΔGint-193 kcal/mol
Surface area74480 Å2

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Components

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


Mass: 42096.953 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P68139
#2: Protein/peptide Lifeact


Mass: 1927.243 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08641*PLUS
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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

ComponentName: Filamentous actin in ADP state / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 7.5
Details: 50mM KCl,1mM MgCl2,0.2mM EGTA, 10mM Imidazole buffer pH 7.5
SpecimenConc.: 0.0002 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 10 mole excess of lifeact was mixed with F-actin and used for sample preparation.
Specimen supportDetails: No prior coating / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 K / Details: blot for 3.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Calibrated magnification: 75000 X / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 120 K / Temperature (min): 100 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 49.2 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 529 / Details: 30

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.15.2_3472refinement
PHENIX1.15.2_3472refinement
EM software
IDNameVersionCategoryDetails
2EPU9image acquisitionRelion
4Gctf1.06CTF correction
7UCSF Chimera9.03model fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionDetails: GCTF for CTF correction / Type: NONE
Helical symmertyAngular rotation/subunit: -166.9 ° / Axial rise/subunit: 27.44 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 111074
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 297584 / Num. of class averages: 100 / Symmetry type: HELICAL
Atomic model buildingB value: 179 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5ONV

5onv


Pdb chain-ID: A / Accession code: 5ONV / Pdb chain residue range: 6-373 / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006115129
ELECTRON MICROSCOPYf_angle_d0.668220521
ELECTRON MICROSCOPYf_chiral_restr0.0462290
ELECTRON MICROSCOPYf_plane_restr0.00382617
ELECTRON MICROSCOPYf_dihedral_angle_d12.37779111

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