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- PDB-7uuw: Cryogenic electron microscopy 3D map of F-actin bound by the Acti... -

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Basic information

Entry
Database: PDB / ID: 7uuw
TitleCryogenic electron microscopy 3D map of F-actin bound by the Actin Binding Domain of alpha-catenin ortholog, HMP1
Components
  • Actin, alpha skeletal muscle
  • Alpha-catenin-like protein hmp-1
KeywordsCELL ADHESION / alpha-catenin / HMP1 / Actin Binding Domain / F-actin / F-actin binding protein / cell-cell junction
Function / homology
Function and homology information


VEGFR2 mediated vascular permeability / embryonic body morphogenesis / cell migration involved in gastrulation / cell-cell adhesion mediated by cadherin / catenin complex / apical junction complex / cortical actin cytoskeleton organization / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration ...VEGFR2 mediated vascular permeability / embryonic body morphogenesis / cell migration involved in gastrulation / cell-cell adhesion mediated by cadherin / catenin complex / apical junction complex / cortical actin cytoskeleton organization / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / regulation of actin cytoskeleton organization / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / beta-catenin binding / cell-cell adhesion / calcium-dependent protein binding / regulation of protein localization / actin filament binding / cell migration / lamellipodium / cell body / hydrolase activity / cadherin binding / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Alpha-catenin / 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 ...Alpha-catenin / 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 / Actin, alpha skeletal muscle / Alpha-catenin-like protein hmp-1
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Caenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsRangarajan, E.S. / Smith, E.W. / Izard, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States) United States
CitationJournal: J Biol Chem / Year: 2023
Title: The nematode α-catenin ortholog, HMP1, has an extended α-helix when bound to actin filaments.
Authors: Erumbi S Rangarajan / Emmanuel W Smith / Tina Izard /
Abstract: The regulation of cell-cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to ...The regulation of cell-cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to filamentous actin at cell-cell junctions. The cadherin-catenin complex plays key roles in cell physiology, organism development, and disease. While mutagenesis of Caenorhabditis elegans cadherin and catenin shows that these proteins are key for embryonic morphogenesis, we know surprisingly little about their structure and attachment to the cytoskeleton. In contrast to mammalian α-catenin that functions as a dimer or monomer, the α-catenin ortholog from C. elegans, HMP1 for humpback, is a monomer. Our cryogenic electron microscopy (cryoEM) structure of HMP1/α-catenin reveals that the amino- and carboxy-terminal domains of HMP1/α-catenin are disordered and not in contact with the remaining HMP1/α-catenin middle domain. Since the carboxy-terminal HMP1/α-catenin domain is the F-actin-binding domain (FABD), this interdomain constellation suggests that HMP1/α-catenin is constitutively active, which we confirm biochemically. Our perhaps most surprising finding, given the high sequence similarity between the mammalian and nematode proteins, is our cryoEM structure of HMP1/α-catenin bound to F-actin. Unlike the structure of mammalian α-catenin bound to F-actin, binding to F-actin seems to allosterically convert a loop region of the HMP1/α-catenin FABD to extend an HMP1/α-catenin FABD α-helix. We use cryoEM and bundling assays to show for the first time how the FABD of HMP1/α-catenin bundles actin in the absence of force. Collectively, our data advance our understanding of α-catenin regulation of cell-cell contacts and additionally aid our understanding of the evolution of multicellularity in metazoans.
History
DepositionApr 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: Alpha-catenin-like protein hmp-1
H: Alpha-catenin-like protein hmp-1
I: Alpha-catenin-like protein hmp-1
K: Alpha-catenin-like protein hmp-1
L: Alpha-catenin-like protein hmp-1
Z: Alpha-catenin-like protein hmp-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)426,69824
Polymers423,98912
Non-polymers2,70912
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "B"
d_3ens_1chain "A"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_1ens_2chain "I"
d_2ens_2chain "H"
d_3ens_2chain "G"
d_4ens_2chain "K"
d_5ens_2chain "L"
d_6ens_2chain "Z"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1THRCYSE1 - 370
d_12ens_1ADPADPF
d_21ens_1THRCYSC1 - 370
d_22ens_1ADPADPD
d_31ens_1THRCYSA1 - 370
d_32ens_1ADPADPB
d_41ens_1THRCYSG1 - 370
d_42ens_1ADPADPH
d_51ens_1THRCYSI1 - 370
d_52ens_1ADPADPJ
d_61ens_1THRCYSK1 - 370
d_62ens_1ADPADPL
d_11ens_2ILEPROO1 - 159
d_21ens_2ILEPRON1 - 159
d_31ens_2ILEPROM1 - 159
d_41ens_2ILEPROP1 - 159
d_51ens_2ILEPROQ1 - 159
d_61ens_2ILEPROR1 - 159

NCS ensembles :
ID
ens_1
ens_2

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Components

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


Mass: 42109.973 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: ACTA1, ACTA / Production host: Escherichia coli (E. coli) / References: UniProt: P68135
#2: Protein
Alpha-catenin-like protein hmp-1 / Protein humpback-1


Mass: 28554.883 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: hmp-1, R13H4.4 / Production host: Escherichia coli (E. coli) / References: UniProt: P90947
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / 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.: 6 / 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

ComponentName: F-actin bound by the Actin Binding Domain of alpha-catenin ortholog, HMP1
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMTrisTris1
31 mMDithiothreitolDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 294.15 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -167 ° / Axial rise/subunit: 27 Å / Axial symmetry: C1
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1100146 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 22.31 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006925296
ELECTRON MICROSCOPYf_angle_d0.718134242
ELECTRON MICROSCOPYf_chiral_restr0.04583912
ELECTRON MICROSCOPYf_plane_restr0.00394338
ELECTRON MICROSCOPYf_dihedral_angle_d15.9213480
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EELECTRON MICROSCOPYNCS constraints0.000701852834337
ens_1d_3EELECTRON MICROSCOPYNCS constraints0.00070415947833
ens_1d_4EELECTRON MICROSCOPYNCS constraints0.000705906350754
ens_1d_5EELECTRON MICROSCOPYNCS constraints0.000703484343975
ens_1d_6EELECTRON MICROSCOPYNCS constraints0.0007060878538
ens_2d_2CELECTRON MICROSCOPYNCS constraints0.000693381819654
ens_2d_3CELECTRON MICROSCOPYNCS constraints0.000693317493966
ens_2d_4CELECTRON MICROSCOPYNCS constraints0.000690743860087
ens_2d_5CELECTRON MICROSCOPYNCS constraints0.0905284469762
ens_2d_6CELECTRON MICROSCOPYNCS constraints0.000696350722949

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