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- PDB-3jbi: MDFF model of the vinculin tail domain bound to F-actin -

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Basic information

Entry
Database: PDB / ID: 3jbi
TitleMDFF model of the vinculin tail domain bound to F-actin
Components
  • Actin, alpha skeletal muscle
  • Vinculin
KeywordsSTRUCTURAL PROTEIN / cytoskeleton / adhesion
Function / homology
Function and homology information


muscle tendon junction / Platelet degranulation / Smooth Muscle Contraction / regulation of protein localization to adherens junction / podosome ring / outer dense plaque of desmosome / inner dense plaque of desmosome / terminal web / epithelial cell-cell adhesion / zonula adherens ...muscle tendon junction / Platelet degranulation / Smooth Muscle Contraction / regulation of protein localization to adherens junction / podosome ring / outer dense plaque of desmosome / inner dense plaque of desmosome / terminal web / epithelial cell-cell adhesion / zonula adherens / dystroglycan binding / vinculin binding / muscle alpha-actinin binding / MAP2K and MAPK activation / alpha-catenin binding / fascia adherens / cell-cell contact zone / costamere / apical junction assembly / regulation of establishment of endothelial barrier / adherens junction assembly / axon extension / protein localization to cell surface / lamellipodium assembly / cytoskeletal motor activator activity / regulation of focal adhesion assembly / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / alpha-actinin binding / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / brush border / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / regulation of cell migration / titin binding / actin filament polymerization / filopodium / cell projection / actin filament / Neutrophil degranulation / morphogenesis of an epithelium / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / sarcolemma / neuromuscular junction / Z disc / beta-catenin binding / calcium-dependent protein binding / actin filament binding / cell-cell junction / actin cytoskeleton / lamellipodium / cell body / scaffold protein binding / mitochondrial inner membrane / cytoskeleton / hydrolase activity / cell adhesion / cadherin binding / protein domain specific binding / focal adhesion / ubiquitin protein ligase binding / calcium ion binding / structural molecule activity / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / protein-containing complex / ATP binding / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Vinculin repeated domain signature. / Vinculin / Vinculin, conserved site / Vinculin family talin-binding region signature. / Vinculin/alpha-catenin / Vinculin family / Alpha-catenin/vinculin-like superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. ...Vinculin repeated domain signature. / Vinculin / Vinculin, conserved site / Vinculin family talin-binding region signature. / Vinculin/alpha-catenin / Vinculin family / Alpha-catenin/vinculin-like superfamily / 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
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Vinculin / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsKim, L.Y. / Thompson, P.M. / Lee, H.T. / Pershad, M. / Campbell, S.L. / Alushin, G.M.
CitationJournal: J Mol Biol / Year: 2016
Title: The Structural Basis of Actin Organization by Vinculin and Metavinculin.
Authors: Laura Y Kim / Peter M Thompson / Hyunna T Lee / Mihir Pershad / Sharon L Campbell / Gregory M Alushin /
Abstract: Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. ...Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies.
History
DepositionSep 2, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2016Group: Database references
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / 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 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
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 / _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_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 / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
V: Vinculin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,8697
Polymers111,9663
Non-polymers9034
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 27.8 Å / Rotation per n subunits: -166.82 °)

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Components

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


Mass: 41862.613 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle / References: UniProt: P68135
#2: Protein Vinculin / / Vt


Mass: 28241.018 Da / Num. of mol.: 1 / Fragment: tail domain (UNP residues 879-1130)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: VCL, VINC1 / Organelle: focal adhesion / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P12003
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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
IDNameTypeParent-ID
1Vinculin tail domain bound to F-actinCOMPLEX0
2skeletal muscle actin1
3Vinculin tail domain, residues 879-10611
Buffer solutionName: 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole / pH: 7 / Details: 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole
SpecimenConc.: 0.0125 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh 1.2 / 1.3 C-flat
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 %
Details: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar Vt was then applied and incubated for 60 seconds. 3 ...Details: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar Vt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of Vt applied. After 60 seconds, 3 microliters of solution was removed, then the grid was blotted for 2 seconds before plunging into liquid ethane (LEICA EM GP).
Method: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar Vt was then applied and incubated for 60 seconds. 3 ...Method: 3 microliters of 0.3 micromolar actin was applied to the grid and incubated for 60 seconds at 25 degrees C. 3 microliters of 10 micromolar Vt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of Vt applied. After 60 seconds, 3 microliters of solution was removed, then the grid was blotted for 2 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: May 22, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 100000 X / Calibrated magnification: 137615 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 1384

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Processing

EM software
IDNameVersionCategory
1CTFFIND3CTF correction
2MDFFmodel fitting
3UCSF Chimeramodel fitting
4EMAN23D reconstruction
5FREALIGN3D reconstruction
6SPARX3D reconstruction
CTF correctionDetails: FREALIGN (per segment)
Helical symmertyAngular rotation/subunit: 166.82 ° / Axial rise/subunit: 27.8 Å / Axial symmetry: C1
3D reconstructionMethod: IHRSR / Resolution: 8.5 Å / Resolution method: FSC 0.143 CUT-OFF / Nominal pixel size: 2.18 Å / Actual pixel size: 2.18 Å / Magnification calibration: catalase crystal
Details: (Helical Details: Single model IHRSR was performed with EMAN2 / SPARX and final reconstruction with FREALIGN (fixed helical parameters).)
Symmetry type: HELICAL
Atomic model building
IDProtocolSpaceDetails
1FLEXIBLE FITREALREFINEMENT PROTOCOL--flexible DETAILS--Components were initially rigid body fit using Chimera, followed by flexible fitting with MDFF.
2FLEXIBLE FITREALREFINEMENT PROTOCOL--flexible DETAILS--Components were initially rigid body fit using Chimera, followed by flexible fitting with MDFF.
Atomic model building

Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-ID
11QKR

1qkr

11QKR1
23J8A

3j8a

23J8A2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms6721 0 56 0 6777

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