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- PDB-8oi8: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring ... -

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Basic information

Entry
Database: PDB / ID: 8oi8
TitleCryo-EM structure of ADP-bound, filamentous beta-actin harboring the R183W mutation
ComponentsActin, cytoplasmic 1
KeywordsSTRUCTURAL PROTEIN / Actin filament / cytoskeletal protein / ATPase
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / nBAF complex / brahma complex / regulation of transepithelial transport / Formation of annular gap junctions / morphogenesis of a polarized epithelium / Formation of the dystrophin-glycoprotein complex (DGC) ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / nBAF complex / brahma complex / regulation of transepithelial transport / Formation of annular gap junctions / morphogenesis of a polarized epithelium / Formation of the dystrophin-glycoprotein complex (DGC) / structural constituent of postsynaptic actin cytoskeleton / GBAF complex / Gap junction degradation / Folding of actin by CCT/TriC / regulation of G0 to G1 transition / protein localization to adherens junction / Cell-extracellular matrix interactions / dense body / postsynaptic actin cytoskeleton / Tat protein binding / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / regulation of double-strand break repair / regulation of nucleotide-excision repair / Adherens junctions interactions / RHOF GTPase cycle / adherens junction assembly / apical protein localization / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / tight junction / SWI/SNF complex / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / positive regulation of T cell differentiation / apical junction complex / regulation of norepinephrine uptake / positive regulation of double-strand break repair / transporter regulator activity / maintenance of blood-brain barrier / nitric-oxide synthase binding / cortical cytoskeleton / NuA4 histone acetyltransferase complex / establishment or maintenance of cell polarity / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / Recycling pathway of L1 / brush border / regulation of G1/S transition of mitotic cell cycle / kinesin binding / EPH-ephrin mediated repulsion of cells / negative regulation of cell differentiation / RHO GTPases Activate WASPs and WAVEs / regulation of synaptic vesicle endocytosis / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / regulation of protein localization to plasma membrane / positive regulation of double-strand break repair via homologous recombination / EPHB-mediated forward signaling / cytoskeleton organization / substantia nigra development / axonogenesis / calyx of Held / nitric-oxide synthase regulator activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / FCGR3A-mediated phagocytosis / actin filament / adherens junction / positive regulation of cell differentiation / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / cell motility / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / DNA Damage Recognition in GG-NER / kinetochore / Regulation of actin dynamics for phagocytic cup formation / B-WICH complex positively regulates rRNA expression / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / platelet aggregation / VEGFA-VEGFR2 Pathway / tau protein binding / Schaffer collateral - CA1 synapse / nuclear matrix / cytoplasmic ribonucleoprotein granule / 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 / cell-cell junction / UCH proteinases / Signaling by BRAF and RAF1 fusions / nucleosome / presynapse / lamellipodium / actin cytoskeleton / Clathrin-mediated endocytosis / HATs acetylate histones / Factors involved in megakaryocyte development and platelet production
Similarity search - Function
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, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.28 Å
AuthorsOosterheert, W. / Blanc, F.E.C. / Roy, A. / Belyy, A. / Hofnagel, O. / Hummer, G. / Bieling, P. / Raunser, S.
Funding support Germany, European Union, 3items
OrganizationGrant numberCountry
Max Planck Society Germany
Alexander von Humboldt Foundation Germany
European Research Council (ERC)856118European Union
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments.
Authors: Wout Oosterheert / Florian E C Blanc / Ankit Roy / Alexander Belyy / Micaela Boiero Sanders / Oliver Hofnagel / Gerhard Hummer / Peter Bieling / Stefan Raunser /
Abstract: The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release ...The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases P through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why P escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated P release and filaments with drastically shortened ADP-P caps. Our results provide the molecular basis for P release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.
History
DepositionMar 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Nov 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Actin, cytoplasmic 1
A: Actin, cytoplasmic 1
B: Actin, cytoplasmic 1
D: Actin, cytoplasmic 1
E: Actin, cytoplasmic 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)211,22115
Polymers208,9635
Non-polymers2,25810
Water10,539585
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"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ALAALAHISHISCA6 - 3716 - 371
d_12ADPADPADPADPCF401
d_21ALAALAHISHISBC6 - 3716 - 371
d_22ADPADPADPADPBJ401
d_31ALAALAHISHISAB6 - 3716 - 371
d_32ADPADPADPADPAH401
d_41ALAALAHISHISDD6 - 3716 - 371
d_42ADPADPADPADPDL401
d_51ALAALAHISHISEE6 - 3716 - 371
d_52ADPADPADPADPEN401

NCS oper:
IDCodeMatrixVector
1given(-0.964520151739, -0.233399926349, -0.123391050205), (0.226007438309, -0.971531230945, 0.0710472035138), (-0.13646067096, 0.0406391643467, 0.989811569746)309.858724101, 220.978695243, -13.2641646899
2given(0.892907583648, 0.449630210801, -0.0234247859805), (-0.450140015426, 0.892596618944, -0.0254016212381), (0.00948754845373, 0.0332257337631, 0.99940284022)-38.4865152081, 75.9709058504, -60.5148201639
3given(-0.962856689403, 0.232690506801, -0.136974901776), (-0.240307899472, -0.969798299055, 0.0417537136155), (-0.123122333974, 0.0731189933883, 0.989694146533)246.026188367, 288.064791549, 35.6738238829
4given(0.888893214479, -0.458113375947, 0.00099399763663), (0.457857439096, 0.888465099373, 0.0315647376143), (-0.0153433607184, -0.0276025718698, 0.999501215261)71.5248099114, -48.6543053678, 60.8597865492

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Components

#1: Protein
Actin, cytoplasmic 1 / Beta-actin


Mass: 41792.633 Da / Num. of mol.: 5 / Mutation: R183W, C272A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB / Plasmid: p2920 pFL_ACTB_R183W_C272A / Cell line (production host): BTI-Tnao38 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P60709, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: 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
#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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 585 / Source method: isolated from a natural source / Formula: H2O
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: single particle reconstruction

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Sample preparation

ComponentName: Actin filament harboring the R183W mutation. / Type: COMPLEX
Details: Actin was purified as monomer from insect cells. It was then polymerized into a filament in vitro.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Cell: BTI-Tnao38
Buffer solutionpH: 7.1
Details: 1x KMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA) supplemented with 0.02% Tween20 (v/v)
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2100 mMpotassium chlorideKCl1
32 mMmagnesium chlorideMgCl21
41 mMEGTA1
50.02 % (v/v)Tween201
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Actin filaments were reconstituted by adding salt to monomeric actin.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Titan Krios G3 microscope was aligned using Sherpa (FEI). Data collected in superresolution mode.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7916
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1crYOLO1.5.8particle selection
2EPUimage acquisition
4CTFFIND4.13CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.20.1-4487-000model refinement
10SPHIRE1.4initial Euler assignment
11RELION3.1.0final Euler assignment
12RELION3.1.0classification
13RELION3.1.03D reconstruction
Image processingDetails: K3 operated in super-resolution mode.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1569882
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1386604 / Details: Refinement from Local searches in RELION. / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: The structure was refined using phenix-real-space refine.
Atomic model buildingAccession code: 8a2t / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 31.83 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003814630
ELECTRON MICROSCOPYf_angle_d0.539419870
ELECTRON MICROSCOPYf_chiral_restr0.04452215
ELECTRON MICROSCOPYf_plane_restr0.00352525
ELECTRON MICROSCOPYf_dihedral_angle_d11.47355395
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints2.34445256493E-11
ens_1d_3AELECTRON MICROSCOPYNCS constraints3.06042325467E-12
ens_1d_4AELECTRON MICROSCOPYNCS constraints8.247743843E-14
ens_1d_5AELECTRON MICROSCOPYNCS constraints2.40769520147E-12

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