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- EMDB-8886: Cryo-EM structure of F-actin complexed with the beta-III-spectrin... -

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Entry
Database: EMDB / ID: EMD-8886
TitleCryo-EM structure of F-actin complexed with the beta-III-spectrin actin-binding domain
Map dataCryo-EM structure of F-actin complexed with the beta-III-spectrin actin-binding domain
Sample
  • Complex: F-actin complexed with the spectrin actin-binding domain
    • Protein or peptide: Actin, cytoplasmic 1
    • Protein or peptide: Spectrin beta chain, non-erythrocytic 2
Keywordsactin binding protein / filament / STRUCTURAL PROTEIN
Function / homology
Function and homology information


cerebellar Purkinje cell layer morphogenesis / structural constituent of synapse / postsynaptic spectrin-associated cytoskeleton / structural constituent of postsynapse / spectrin / positive regulation of norepinephrine uptake / paranodal junction / regulation of postsynaptic specialization assembly / cellular response to cytochalasin B / bBAF complex ...cerebellar Purkinje cell layer morphogenesis / structural constituent of synapse / postsynaptic spectrin-associated cytoskeleton / structural constituent of postsynapse / spectrin / positive regulation of norepinephrine uptake / paranodal junction / regulation of postsynaptic specialization assembly / cellular response to cytochalasin B / bBAF complex / npBAF complex / regulation of transepithelial transport / nBAF complex / brahma complex / morphogenesis of a polarized epithelium / protein localization to adherens junction / postsynaptic actin cytoskeleton / structural constituent of postsynaptic actin cytoskeleton / GBAF complex / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / Tat protein binding / Gap junction degradation / regulation of G0 to G1 transition / Folding of actin by CCT/TriC / dense body / Cell-extracellular matrix interactions / actin filament capping / regulation of nucleotide-excision repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / apical protein localization / regulation of double-strand break repair / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / tight junction / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / 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 / regulation of norepinephrine uptake / apical junction complex / transporter regulator activity / cortical actin cytoskeleton / positive regulation of double-strand break repair / nitric-oxide synthase binding / maintenance of blood-brain barrier / NuA4 histone acetyltransferase complex / establishment or maintenance of cell polarity / cortical cytoskeleton / parallel fiber to Purkinje cell synapse / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / adult behavior / Recycling pathway of L1 / regulation of G1/S transition of mitotic cell cycle / brush border / regulation of synaptic vesicle endocytosis / kinesin binding / EPH-ephrin mediated repulsion of cells / negative regulation of cell differentiation / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / synapse assembly / vesicle-mediated transport / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / NCAM signaling for neurite out-growth / calyx of Held / MHC class II antigen presentation / axonogenesis / cell projection / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / adherens junction / actin filament / positive regulation of cell differentiation / FCGR3A-mediated phagocytosis / cell motility / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / DNA Damage Recognition in GG-NER / multicellular organism growth / B-WICH complex positively regulates rRNA expression / kinetochore / phospholipid binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / tau protein 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)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 7.0 Å
AuthorsWang F / Orlova A
Funding support United States, 3 items
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, 2017-
Header (metadata) releaseSep 6, 2017-
Map releaseNov 22, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.95
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.95
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  • Surface view with fitted model
  • Atomic models: PDB-6anu
  • Surface level: 0.95
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6anu
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8886.png

Downloads & links

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Map

FileDownload / File: emd_8886.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of F-actin complexed with the beta-III-spectrin actin-binding domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 200 pix.
= 210. Å		1.05 Å/pix.
x 200 pix.
= 210. Å		1.05 Å/pix.
x 200 pix.
= 210. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.95 / Movie #1: 0.95
Minimum - Maximum-0.6216059 - 2.428842
Average (Standard dev.)0.12115186 (±0.32677585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 209.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.6222.4290.121

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Supplemental data

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Sample components

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Entire : F-actin complexed with the spectrin actin-binding domain

EntireName: F-actin complexed with the spectrin actin-binding domain
Components
  • Complex: F-actin complexed with the spectrin actin-binding domain
    • Protein or peptide: Actin, cytoplasmic 1
    • Protein or peptide: Spectrin beta chain, non-erythrocytic 2

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Supramolecule #1: F-actin complexed with the spectrin actin-binding domain

SupramoleculeName: F-actin complexed with the spectrin actin-binding domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Actin, cytoplasmic 1

MacromoleculeName: Actin, cytoplasmic 1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.78266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: Spectrin beta chain, non-erythrocytic 2

MacromoleculeName: Spectrin beta chain, non-erythrocytic 2 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.857141 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSTLSPTDF DSLEIQGQYS DINNRWDLPD SDWDNDSSSA RLFERSRIKA LADEREAVQK KTFTKWVNSH LARVTCRVGD LYSDLRDGR NLLRLLEVLS GEILPKPTKG RMRIHCLENV DKALQFLKEQ KVHLENMGSH DIVDGNHRLT LGLVWTIILR F QIQDISVE ...String:
MSSTLSPTDF DSLEIQGQYS DINNRWDLPD SDWDNDSSSA RLFERSRIKA LADEREAVQK KTFTKWVNSH LARVTCRVGD LYSDLRDGR NLLRLLEVLS GEILPKPTKG RMRIHCLENV DKALQFLKEQ KVHLENMGSH DIVDGNHRLT LGLVWTIILR F QIQDISVE TEDNKEKKSA KDALLLWCQM KTAGYPNVNV HNFTTSWRDG LAFNAIVHKH RPDLLDFESL KKCNAHYNLQ NA FNLAEKE LGLTKPLDPE DVNVDQPDEK SIITYVATYY HYFSKMK

UniProtKB: Spectrin beta chain, non-erythrocytic 2

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
StainingType: NEGATIVE / Material: negative stain
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 3.0 sec. / Average electron dose: 20.0 e/Å2
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).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.25 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.87 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: SPIDER / Details: model-map FSC 0.38 cut-off / Number images used: 12443
Startup modelType of model: OTHER / Details: low resolution pure actin filament map
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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