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- PDB-3jbk: Cryo-EM reconstruction of the metavinculin-actin interface -

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Basic information

Entry
Database: PDB / ID: 3jbk
TitleCryo-EM reconstruction of the metavinculin-actin interface
Components
  • Actin, alpha skeletal muscle
  • Metavinculin
KeywordsSTRUCTURAL PROTEIN / actin / metavinculin / vinculin / cell migration / adhesion / mechanosensation / cytoskeleton
Function / homology
Function and homology information


regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / cell-substrate junction / epithelial cell-cell adhesion / zonula adherens / dystroglycan binding / alpha-catenin binding ...regulation of protein localization to adherens junction / outer dense plaque of desmosome / inner dense plaque of desmosome / podosome ring / terminal web / cell-substrate junction / epithelial cell-cell adhesion / zonula adherens / dystroglycan binding / alpha-catenin binding / fascia adherens / cell-cell contact zone / apical junction assembly / costamere / regulation of establishment of endothelial barrier / adherens junction assembly / axon extension / protein localization to cell surface / lamellipodium assembly / regulation of focal adhesion assembly / cytoskeletal motor activator activity / myosin heavy chain binding / maintenance of blood-brain barrier / tropomyosin binding / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle / brush border / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / Smooth Muscle Contraction / stress fiber / skeletal muscle fiber development / titin binding / actin filament polymerization / negative regulation of cell migration / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / cell-matrix adhesion / morphogenesis of an epithelium / cell projection / filopodium / adherens junction / actin filament / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / sarcolemma / beta-catenin binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / platelet aggregation / specific granule lumen / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / calcium-dependent protein binding / Signaling by BRAF and RAF1 fusions / Signaling by ALK fusions and activated point mutants / cell-cell junction / Platelet degranulation / extracellular vesicle / lamellipodium / actin binding / cell body / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / secretory granule lumen / molecular adaptor activity / ficolin-1-rich granule lumen / cytoskeleton / hydrolase activity / cell adhesion / cadherin binding / protein domain specific binding / membrane raft / focal adhesion / ubiquitin protein ligase binding / calcium ion binding / Neutrophil degranulation / positive regulation of gene expression / structural molecule activity / magnesium ion binding / protein-containing complex / extracellular exosome / extracellular region / ATP binding / identical protein binding / plasma membrane / cytosol / 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 speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.2 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 3, 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_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.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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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
M: Metavinculin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,5077
Polymers113,6043
Non-polymers9034
Water00
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: -166.75 °)

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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 Metavinculin / MVt


Mass: 29878.275 Da / Num. of mol.: 1 / Fragment: tail domain (UNP residues 858-1129)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCL / Organelle: focal adhesion / Plasmid: 2HR-T / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P18206
#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


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
IDNameTypeDetailsParent-ID
1Metavinculin tail domain bound to F-actinCOMPLEXOne metavinculin tail domain per actin protomer0
2skeletal muscle actin1
3Metavinculin tail domain residues 858-11291
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 MVt was then applied and incubated for 60 seconds. ...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 MVt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of MVt 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 MVt 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 MVt was then applied and incubated for 60 seconds. 3 microliters of solution was removed, then an additional 3 microliters of MVt 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

MicroscopyModel: FEI TECNAI 20 / Date: Oct 10, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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: 671

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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: 166.75 ° / Axial rise/subunit: 27.8 Å / Axial symmetry: C1
3D reconstructionMethod: IHRSR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Nominal pixel size: 2.18 Å / Actual pixel size: 2.18 Å
Details: (Helical Details: Multi-model IHRSR was performed using EMAN2 / SPARX to select for bound segments, followed by single model IHRSR with EMAN2 / SPARX and final reconstruction with FREALIGN ...Details: (Helical Details: Multi-model IHRSR was performed using EMAN2 / SPARX to select for bound segments, followed by single model IHRSR 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. atoms6720 0 56 0 6776

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