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- PDB-6anu: Cryo-EM structure of F-actin complexed with the beta-III-spectrin... -

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Basic information

Entry
Database: PDB / ID: 6anu
TitleCryo-EM structure of F-actin complexed with the beta-III-spectrin actin-binding domain
Components
  • Actin, cytoplasmic 1
  • Spectrin beta chain, non-erythrocytic 2
KeywordsSTRUCTURAL PROTEIN / actin binding protein / filament
Function / homology
Function and homology information


cerebellar Purkinje cell layer morphogenesis / structural constituent of synapse / postsynaptic spectrin-associated cytoskeleton / structural constituent of postsynapse / regulation of postsynaptic specialization assembly / spectrin / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / paranodal junction ...cerebellar Purkinje cell layer morphogenesis / structural constituent of synapse / postsynaptic spectrin-associated cytoskeleton / structural constituent of postsynapse / regulation of postsynaptic specialization assembly / spectrin / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / paranodal junction / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / npBAF complex / protein localization to adherens junction / nBAF complex / brahma complex / Tat protein binding / postsynaptic actin cytoskeleton / structural constituent of postsynaptic actin cytoskeleton / GBAF complex / Formation of annular gap junctions / regulation of G0 to G1 transition / Gap junction degradation / dense body / Cell-extracellular matrix interactions / Folding of actin by CCT/TriC / apical protein localization / regulation of double-strand break repair / actin filament capping / adherens junction assembly / regulation of nucleotide-excision repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / RHOF GTPase cycle / Adherens junctions interactions / tight junction / Interaction between L1 and Ankyrins / Sensory processing of sound by outer hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / SWI/SNF complex / regulation of norepinephrine uptake / parallel fiber to Purkinje cell synapse / regulation of synaptic vesicle endocytosis / positive regulation of double-strand break repair / apical junction complex / positive regulation of T cell differentiation / regulation of cyclin-dependent protein serine/threonine kinase activity / establishment or maintenance of cell polarity / cortical actin cytoskeleton / maintenance of blood-brain barrier / cortical cytoskeleton / NuA4 histone acetyltransferase complex / positive regulation of stem cell population maintenance / adult behavior / nitric-oxide synthase binding / Regulation of MITF-M-dependent genes involved in pigmentation / regulation of G1/S transition of mitotic cell cycle / Recycling pathway of L1 / brush border / kinesin binding / negative regulation of cell differentiation / calyx of Held / EPH-ephrin mediated repulsion of cells / regulation of protein localization to plasma membrane / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / positive regulation of double-strand break repair via homologous recombination / RHO GTPases activate IQGAPs / COPI-mediated anterograde transport / vesicle-mediated transport / synapse assembly / EPHB-mediated forward signaling / MHC class II antigen presentation / substantia nigra development / NCAM signaling for neurite out-growth / axonogenesis / negative regulation of protein binding / cell projection / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / cell motility / Translocation of SLC2A4 (GLUT4) to the plasma membrane / actin filament / RHO GTPases Activate Formins / positive regulation of cell differentiation / adherens junction / FCGR3A-mediated phagocytosis / regulation of transmembrane transporter activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Signaling by high-kinase activity BRAF mutants / DNA Damage Recognition in GG-NER / MAP2K and MAPK activation / tau protein binding / B-WICH complex positively regulates rRNA expression / multicellular organism growth / Schaffer collateral - CA1 synapse / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / phospholipid binding
Similarity search - Function
Pleckstrin homology domain, spectrin-type / Pleckstrin homology domain 9 / Pleckstrin homology domain / Spectrin, beta subunit / Spectrin repeat / Spectrin repeat / Spectrin/alpha-actinin / Spectrin repeats / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. ...Pleckstrin homology domain, spectrin-type / Pleckstrin homology domain 9 / Pleckstrin homology domain / Spectrin, beta subunit / Spectrin repeat / Spectrin repeat / Spectrin/alpha-actinin / Spectrin repeats / 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 / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / ATPase, nucleotide binding domain / PH-like domain superfamily
Similarity search - Domain/homology
Spectrin beta chain, non-erythrocytic 2 / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / negative staining / cryo EM / Resolution: 7 Å
AuthorsWang, F. / Orlova, A. / Avery, A.W. / Hays, T.S. / Egelman, E.H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM81303 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM44757 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)AG32961 United States
CitationJournal: Nat Commun / Year: 2017
Title: Structural basis for high-affinity actin binding revealed by a β-III-spectrin SCA5 missense mutation.
Authors: Adam W Avery / Michael E Fealey / Fengbin Wang / Albina Orlova / Andrew R Thompson / David D Thomas / Thomas S Hays / Edward H Egelman /
Abstract: Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline ...Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which β-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.
History
DepositionAug 14, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 22, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
F: Actin, cytoplasmic 1
A: Actin, cytoplasmic 1
B: Actin, cytoplasmic 1
C: Actin, cytoplasmic 1
D: Actin, cytoplasmic 1
E: Actin, cytoplasmic 1
f: Spectrin beta chain, non-erythrocytic 2
a: Spectrin beta chain, non-erythrocytic 2
b: Spectrin beta chain, non-erythrocytic 2
c: Spectrin beta chain, non-erythrocytic 2
d: Spectrin beta chain, non-erythrocytic 2
e: Spectrin beta chain, non-erythrocytic 2


Theoretical massNumber of molelcules
Total (without water)447,83912
Polymers447,83912
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, helical filament was observed by negative staining and Cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36340 Å2
ΔGint-117 kcal/mol
Surface area108660 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 20 / Rise per n subunits: 27.25 Å / Rotation per n subunits: -166.87 °)
DetailsTHE ASSEMBLY REPRESENTED IN THIS ENTRY HAS REGULAR HELICAL SYMMETRY WITH THE FOLLOWING PARAMETERS: ROTATION PER SUBUNIT (TWIST) = -166.87 DEGREES RISE PER SUBUNIT (HEIGHT) = 27.25 ANGSTROMS

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Components

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


Mass: 41782.660 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB / Production host: Escherichia coli (E. coli) / References: UniProt: P60709
#2: Protein
Spectrin beta chain, non-erythrocytic 2 / Beta-III spectrin / Spinocerebellar ataxia 5 protein


Mass: 32857.141 Da / Num. of mol.: 6 / Mutation: L253P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPTBN2, KIAA0302, SCA5 / Production host: Escherichia coli (E. coli) / References: UniProt: O15020

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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 complexed with the spectrin actin-binding domain
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES
EM stainingType: NEGATIVE / Material: negative stain
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 FIELD
Image recordingAverage exposure time: 3 sec. / Electron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)
Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the ...Details: Images were stored containing seven parts, where each part represented a set of frames corresponding to a dose of ~20 electrons per Angstrom^2. The full dose image stack was used for the estimation of the CTF as well as for boxing filaments. Only the first two parts were used for the reconstruction (~5 electrons per Angstrom^2).
Image scansMovie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: dev_2471: / Classification: refinement
EM software
IDNameCategory
1EMAN2particle selection
2EPUimage acquisition
4CTFFIND3CTF correction
7Rosettamodel fitting
9SPIDERinitial Euler assignment
10SPIDERfinal Euler assignment
12SPIDER3D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.87 ° / Axial rise/subunit: 27.25 Å / Axial symmetry: C1
3D reconstructionResolution: 7 Å / Resolution method: OTHER / Num. of particles: 12443 / Algorithm: BACK PROJECTION / Details: model-map FSC 0.38 cut-off / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00648396
ELECTRON MICROSCOPYf_angle_d1.00387534
ELECTRON MICROSCOPYf_dihedral_angle_d7.67219314
ELECTRON MICROSCOPYf_chiral_restr0.0553714
ELECTRON MICROSCOPYf_plane_restr0.0067344

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