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- PDB-6m5g: F-actin-Utrophin complex -

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Basic information

Entry
Database: PDB / ID: 6m5g
TitleF-actin-Utrophin complex
Components
  • Actin, alpha skeletal muscle
  • Utrophin
KeywordsCONTRACTILE PROTEIN / Utrophin / N-terminus actin binding domain / calponin homology / F-actin / F-actin marker protein
Function / homology
Function and homology information


synaptic signaling / dystrophin-associated glycoprotein complex / Striated Muscle Contraction / vinculin binding / EGR2 and SOX10-mediated initiation of Schwann cell myelination / regulation of sodium ion transmembrane transport / Formation of the dystrophin-glycoprotein complex (DGC) / filopodium membrane / positive regulation of cell-matrix adhesion / muscle organ development ...synaptic signaling / dystrophin-associated glycoprotein complex / Striated Muscle Contraction / vinculin binding / EGR2 and SOX10-mediated initiation of Schwann cell myelination / regulation of sodium ion transmembrane transport / Formation of the dystrophin-glycoprotein complex (DGC) / filopodium membrane / positive regulation of cell-matrix adhesion / muscle organ development / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber / muscle contraction / filopodium / actin filament / neuromuscular junction / sarcolemma / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / integrin binding / actin cytoskeleton / actin binding / postsynaptic membrane / cytoskeleton / hydrolase activity / cilium / ciliary basal body / protein kinase binding / protein-containing complex / extracellular exosome / zinc ion binding / nucleoplasm / ATP binding / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Dystrophin/utrophin / EF-hand domain, type 1 / EF-hand domain, type 2 / EF hand / EF-hand / : / Calponin-like domain / Actin-binding Protein, T-fimbrin; domain 1 / Spectrin repeat / Spectrin repeat ...Dystrophin/utrophin / EF-hand domain, type 1 / EF-hand domain, type 2 / EF hand / EF-hand / : / Calponin-like domain / Actin-binding Protein, T-fimbrin; domain 1 / Spectrin repeat / Spectrin repeat / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Spectrin/alpha-actinin / Spectrin repeats / Calponin homology domain / Zinc finger ZZ-type signature. / Calponin homology (CH) domain / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / WW/rsp5/WWP domain signature. / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW 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 / EF-hand domain pair / Alpha-Beta Complex / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Utrophin / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKumari, A. / Ragunath, V.K. / Sirajuddin, M.
Funding support India, 2items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/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
DepositionMar 10, 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 / Category: citation / Item: _citation.journal_volume
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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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-30085
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
F: Utrophin
G: Utrophin
H: Utrophin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)305,05818
Polymers302,8018
Non-polymers2,25810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Co-sedimentation assay
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24990 Å2
ΔGint-193 kcal/mol
Surface area83590 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 Utrophin / Dystrophin-related protein 1 / DRP-1


Mass: 30771.949 Da / Num. of mol.: 3 / Fragment: calponin domain 1, calponin domain 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UTRN, DMDL, DRP1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P46939
#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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Filamentous actin in ADP stateCOMPLEX#1-#20MULTIPLE SOURCES
2ActinCOMPLEX#11NATURALFilamentous actin
3utrophinCOMPLEX#21RECOMBINANTactin binding domain of utrophin
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 0.0002 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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 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 / Nominal magnification: 59000 X / Nominal defocus max: 4735 nm / Nominal defocus min: 1250 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: 42.6 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 765 / Details: 20

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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 Chimeramodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionDetails: GCTF for CTF correction / Type: NONE
Helical symmertyAngular rotation/subunit: -166.6 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 259938
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149660 / Symmetry type: HELICAL
Atomic model buildingB value: 177 / 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_d0.010417729
ELECTRON MICROSCOPYf_angle_d0.798224044
ELECTRON MICROSCOPYf_chiral_restr0.05032691
ELECTRON MICROSCOPYf_plane_restr0.00423070
ELECTRON MICROSCOPYf_dihedral_angle_d11.590610665

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