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

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Basic information

Entry
Database: PDB / ID: 8oid
TitleCryo-EM structure of ADP-bound, filamentous beta-actin harboring the N111S mutation
ComponentsActin, cytoplasmic 1, N-terminally processed
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 / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / postsynaptic actin cytoskeleton organization / regulation of transepithelial transport / brahma complex / nBAF complex / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / Formation of annular gap junctions / GBAF complex / Gap junction degradation / postsynaptic actin cytoskeleton / protein localization to adherens junction / regulation of G0 to G1 transition / dense body / Cell-extracellular matrix interactions / Tat protein binding / Folding of actin by CCT/TriC / regulation of double-strand break repair / regulation of nucleotide-excision repair / RSC-type complex / apical protein localization / Prefoldin mediated transfer of substrate to CCT/TriC / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / tight junction / Sensory processing of sound by outer hair cells of the cochlea / SWI/SNF complex / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / regulation of norepinephrine uptake / positive regulation of double-strand break repair / positive regulation of T cell differentiation / NuA4 histone acetyltransferase complex / regulation of synaptic vesicle endocytosis / apical junction complex / maintenance of blood-brain barrier / establishment or maintenance of cell polarity / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / positive regulation of stem cell population maintenance / nitric-oxide synthase binding / Recycling pathway of L1 / regulation of cyclin-dependent protein serine/threonine kinase activity / regulation of G1/S transition of mitotic cell cycle / negative regulation of cell differentiation / brush border / kinesin binding / calyx of Held / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / positive regulation of myoblast differentiation / regulation of protein localization to plasma membrane / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / negative regulation of protein binding / actin filament / cell motility / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of transmembrane transporter activity / positive regulation of cell differentiation / FCGR3A-mediated phagocytosis / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA Damage Recognition in GG-NER / tau protein binding / Signaling by high-kinase activity BRAF mutants / Schaffer collateral - CA1 synapse / MAP2K and MAPK activation / B-WICH complex positively regulates rRNA expression / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / kinetochore / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / nuclear matrix / VEGFA-VEGFR2 Pathway / UCH proteinases / 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 / nucleosome / cell-cell junction / Signaling by BRAF and RAF1 fusions / actin cytoskeleton / presynapse / lamellipodium / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / blood microparticle / regulation of apoptotic process
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: 2.3 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 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Source and taxonomy / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / entity / entity_src_gen / struct_ncs_dom_lim / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.year / _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_gene / _struct_ncs_dom_lim.beg_auth_asym_id / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_auth_seq_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_asym_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_auth_seq_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq_dif.pdbx_seq_db_accession_code
Revision 1.2Sep 27, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.pdbx_database_id_DOI / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.3Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.4Nov 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, N-terminally processed
A: Actin, cytoplasmic 1, N-terminally processed
B: Actin, cytoplasmic 1, N-terminally processed
D: Actin, cytoplasmic 1, N-terminally processed
E: Actin, cytoplasmic 1, N-terminally processed
hetero molecules


Theoretical massNumber of molelcules
Total (without water)210,94015
Polymers208,6835
Non-polymers2,25810
Water11,097616
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_1(chain "B" and (resid 6 through 160 or resid 162 through 375 or resid 376 through 377))
d_2ens_1(chain "C" and (resid 6 through 160 or resid 162 through 375 or resid 376 through 377))
d_3ens_1(chain "A" and (resid 6 through 160 or resid 162 through 375 or resid 376 through 377))
d_4ens_1(chain "D" and (resid 6 through 160 or resid 162 through 375 or resid 376 through 377))
d_5ens_1(chain "E" and (resid 6 through 160 or resid 162 through 375 or resid 376 through 377))

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_11ALAALATHRTHRBC6 - 1606 - 160
d_12THRTHRPHEPHEBC162 - 375162 - 375
d_13ADPADPADPADPBJ401
d_21ALAALATHRTHRCA6 - 1606 - 160
d_22THRTHRPHEPHECA162 - 375162 - 375
d_23ADPADPADPADPCF401
d_31ALAALATHRTHRAB6 - 1606 - 160
d_32THRTHRPHEPHEAB162 - 375162 - 375
d_33ADPADPADPADPAH401
d_41ALAALATHRTHRDD6 - 1606 - 160
d_42THRTHRPHEPHEDD162 - 375162 - 375
d_43ADPADPADPADPDL401
d_51ALAALATHRTHREE6 - 1606 - 160
d_52THRTHRPHEPHEEE162 - 375162 - 375
d_53ADPADPADPADPEN401

NCS oper:
IDCodeMatrixVector
1given(-0.962612549378, 0.228009418417, -0.146249050911), (-0.238302515526, -0.969517806885, 0.0569836224519), (-0.128798256483, 0.0897046668066, 0.987605225725)247.711208929, 285.608421535, 34.2799331083
2given(-0.962839055733, -0.237900736544, -0.127844406633), (0.227589897339, -0.969571964293, 0.0901833947415), (-0.145409048496, 0.0577359992546, 0.987685558771)310.805691961, 217.487882682, -14.2920310895
3given(0.891310618266, -0.453302147553, 0.00908541634436), (0.452725272878, 0.890906176814, 0.0364144396281), (-0.0246009972255, -0.028343379105, 0.99929547372)69.3942387951, -48.4233933871, 62.0779640179
4given(-0.750155225382, 0.644607511091, -0.147473029662), (-0.653779964153, -0.756441596544, 0.0191799241655), (-0.0991912108253, 0.110802832381, 0.988880091837)160.729086178, 319.475163973, 82.3915480885

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Components

#1: Protein
Actin, cytoplasmic 1, N-terminally processed /


Mass: 41736.590 Da / Num. of mol.: 5 / Mutation: N111S, C272A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB / Plasmid: p2925 pFL_ACTB_N111S_C272A / Cell line (production host): BTI-Tnao38 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P60709
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine 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 / Water


Mass: 18.015 Da / Num. of mol.: 616 / 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 N111S mutation. / Type: COMPLEX
Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. ...Details: Beta-actin was expressed as fusion protein, with thymosin beta4 and a deca-His-tag fused to the actin C-terminus. During the purification, thymosin beta-4 and the deca-His-tag were removed. 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.: 2.5 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-PROPANE / 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 FIELDBright-field microscopy / 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: 9516
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
9SPHIRE1.4initial Euler assignment
10RELION3.1.0final Euler assignment
11RELION3.1.0classification
12RELION3.1.03D reconstruction
13PHENIX1.20.1-4487-000model refinement
Image processingDetails: K3 operated in super-resolution mode.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2001281 / Details: crYOLO in filament mode.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1756928
Details: The final refinement was performed from local searches in RELION.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: chain C of pdb 8A2T (including all water molecules) was fit in the central actin subunit of the density map. After substitution of all alpha-actin specific amino-acids to the corresponding ...Details: chain C of pdb 8A2T (including all water molecules) was fit in the central actin subunit of the density map. After substitution of all alpha-actin specific amino-acids to the corresponding beta-actin residues, introducing the N111S mutation, and further manual model building in Coot, the resulting model was fitted in four more actin subunits (chains A, B, D, E) in the density map. The filament was modeled as a pentamer to capture the full interaction interface of the central subunit with its four neighboring subunits. All water molecules were first manually built, inspected and adjusted in the central subunit, and were then copied to the other chains with non-crystallographic symmetry (NCS). Because the local resolution was worse at the periphery of the reconstruction, we removed water molecules that displayed poor corresponding cryo-EM density in the non-central actin chains. The model was refined in Phenix real-space refine with NCS restraints but without Ramachandran and rotamer restraints.
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: 30.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003714920
ELECTRON MICROSCOPYf_angle_d0.576420250
ELECTRON MICROSCOPYf_chiral_restr0.04412245
ELECTRON MICROSCOPYf_plane_restr0.00412585
ELECTRON MICROSCOPYf_dihedral_angle_d13.04535545
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints4.92018537194E-13
ens_1d_3CELECTRON MICROSCOPYNCS constraints5.09994634163E-13
ens_1d_4CELECTRON MICROSCOPYNCS constraints5.09314316774E-13
ens_1d_5CELECTRON MICROSCOPYNCS constraints4.70681364784E-12

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