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- PDB-6vec: Cryo-EM structure of F-actin/Plastin2-ABD2 complex -

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Basic information

Entry
Database: PDB / ID: 6vec
TitleCryo-EM structure of F-actin/Plastin2-ABD2 complex
Components
  • Actin, alpha skeletal muscle
  • LCP1
KeywordsPROTEIN FIBRIL / F-actin / Plastin 2 / Helical reconstruction
Function / homology
Function and homology information


actin filament network formation / actin crosslink formation / T cell activation involved in immune response / podosome / positive regulation of podosome assembly / cortical actin cytoskeleton organization / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / myosin heavy chain binding ...actin filament network formation / actin crosslink formation / T cell activation involved in immune response / podosome / positive regulation of podosome assembly / cortical actin cytoskeleton organization / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / myosin heavy chain binding / regulation of intracellular protein transport / troponin I binding / filamentous actin / actin filament bundle / glial cell projection / phagocytic cup / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / extracellular matrix disassembly / animal organ regeneration / skeletal muscle fiber development / stress fiber / titin binding / ruffle / protein kinase A signaling / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ruffle membrane / calcium-dependent protein binding / actin filament binding / cell migration / actin cytoskeleton / integrin binding / GTPase binding / lamellipodium / cell junction / actin binding / cell body / hydrolase activity / protein domain specific binding / focal adhesion / calcium ion binding / positive regulation of gene expression / perinuclear region of cytoplasm / magnesium ion binding / extracellular space / extracellular exosome / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Plastin-2/3 / Fimbrin/Plastin / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. ...Plastin-2/3 / Fimbrin/Plastin / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / EF-hand domain pair / EF-hand, calcium binding motif / ATPase, nucleotide binding domain / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Plastin-2 / Actin, alpha skeletal muscle / Epididymis secretory protein Li 37
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZheng, W. / Kudryashov, D.S. / Egelman, E.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122510 United States
CitationJournal: Bone Res / Year: 2020
Title: Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.
Authors: Christopher L Schwebach / Elena Kudryashova / Weili Zheng / Matthew Orchard / Harper Smith / Lucas A Runyan / Edward H Egelman / Dmitri S Kudryashov /
Abstract: Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent ...Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent osteoporosis are understood. Of the over 20 identified osteoporosis-linked PLS3 mutations, we investigated all five that are expected to produce full-length protein. One of the mutations distorted an actin-binding loop in the second actin-binding domain of PLS3 and abolished F-actin bundling as revealed by cryo-EM reconstruction and protein interaction assays. Surprisingly, the remaining four mutants fully retained F-actin bundling ability. However, they displayed defects in Ca sensitivity: two of the mutants lost the ability to be inhibited by Ca, while the other two became hypersensitive to Ca. Each group of the mutants with similar biochemical properties showed highly characteristic cellular behavior. Wild-type PLS3 was distributed between lamellipodia and focal adhesions. In striking contrast, the Ca-hyposensitive mutants were not found at the leading edge but localized exclusively at focal adhesions/stress fibers, which displayed reinforced morphology. Consistently, the Ca-hypersensitive PLS3 mutants were restricted to lamellipodia, while chelation of Ca caused their redistribution to focal adhesions. Finally, the bundling-deficient mutant failed to co-localize with any F-actin structures in cells despite a preserved F-actin binding through a non-mutation-bearing actin-binding domain. Our findings revealed that severe osteoporosis can be caused by a mutational disruption of the Ca-controlled PLS3's cycling between adhesion complexes and the leading edge. Integration of the structural, biochemical, and cell biology insights enabled us to propose a molecular mechanism of plastin activity regulation by Ca.
History
DepositionDec 31, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Structure summary / Category: citation / citation_author / entity
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _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 / _entity.pdbx_description
Revision 1.2Mar 6, 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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  • Superimposition on EM map
  • EMDB-21155
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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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: Actin, alpha skeletal muscle
G: Actin, alpha skeletal muscle
H: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
a: LCP1
b: LCP1
c: LCP1
d: LCP1
e: LCP1
f: LCP1
g: LCP1
h: LCP1
i: LCP1
j: LCP1
k: LCP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)995,85344
Polymers990,88622
Non-polymers4,96722
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area65210 Å2
ΔGint-482 kcal/mol
Surface area280790 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 11 / Rise per n subunits: 28 Å / Rotation per n subunits: -166.5 °)

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Components

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


Mass: 42096.953 Da / Num. of mol.: 11 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein
LCP1


Mass: 47983.633 Da / Num. of mol.: 11 / Fragment: UNP residues 385-625
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HEL-S-37
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: V9HWJ7, UniProt: P13796*PLUS
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg
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 of Plastin2-ABD2 with F-actinCOMPLEX#1-#20MULTIPLE SOURCES
2F-actinActinCOMPLEX#11NATURAL
3Plastin2-ABD2COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Oryctolagus cuniculus (rabbit)9986
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2EPUimage acquisition
4RELION3CTF correction
7RosettaEMmodel fitting
9RELIONinitial Euler assignment
12RELION3D reconstruction
13Cootmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.5 ° / Axial rise/subunit: 28 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 248184
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124092 / Symmetry type: HELICAL
Atomic model buildingPDB-ID: 5ONV

5onv


Accession code: 5ONV / Source name: PDB / Type: experimental model

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