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- EMDB-16888: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring ... -

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Entry
Database: EMDB / ID: EMD-16888
TitleCryo-EM structure of ADP-bound, filamentous beta-actin harboring the R183W mutation
Map dataSharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the R183W mutation.
Sample
  • Complex: Actin filament harboring the R183W mutation.
    • Protein or peptide: Actin, cytoplasmic 1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsActin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / npBAF complex / protein localization to adherens junction / nBAF complex / brahma complex ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / 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 / 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 / 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 / maintenance of blood-brain barrier / cortical cytoskeleton / NuA4 histone acetyltransferase complex / positive regulation of stem cell population maintenance / 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 / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / negative regulation of protein binding / 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 / Schaffer collateral - CA1 synapse / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / platelet aggregation / kinetochore / VEGFA-VEGFR2 Pathway / 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 / UCH proteinases / Signaling by BRAF and RAF1 fusions / cell-cell junction / nucleosome / actin cytoskeleton / lamellipodium / presynapse / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones
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
Actin, cytoplasmic 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.28 Å
AuthorsOosterheert W / Blanc FEC / Roy A / Belyy A / Hofnagel O / Hummer G / Bieling P / Raunser S
Funding support Germany, European Union, 3 items
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, 2023-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16888.png

Downloads & links

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Map

FileDownload / File: emd_16888.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the R183W mutation.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.7 Å/pix.
x 384 pix.
= 266.88 Å		0.7 Å/pix.
x 384 pix.
= 266.88 Å		0.7 Å/pix.
x 384 pix.
= 266.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.695 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.038
Minimum - Maximum-0.12874886 - 0.36157265
Average (Standard dev.)0.00017974104 (±0.0063069593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16888_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D refined, unsharpened cryo-EM density map of filamentous...

Fileemd_16888_additional_1.map
Annotation3D refined, unsharpened cryo-EM density map of filamentous beta-actin harboring the R183W mutation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the refinement of filamentous...

Fileemd_16888_half_map_1.map
AnnotationHalf map 1 of the refinement of filamentous beta-actin harboring the R183W mutation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the refinement of filamentous...

Fileemd_16888_half_map_2.map
AnnotationHalf map 2 of the refinement of filamentous beta-actin harboring the R183W mutation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actin filament harboring the R183W mutation.

EntireName: Actin filament harboring the R183W mutation.
Components
  • Complex: Actin filament harboring the R183W mutation.
    • Protein or peptide: Actin, cytoplasmic 1
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Actin filament harboring the R183W mutation.

SupramoleculeName: Actin filament harboring the R183W mutation. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Actin was purified as monomer from insect cells. It was then polymerized into a filament in vitro.
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: 5 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.792633 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGWDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESAGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 585 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration2.3 mg/mL
BufferpH: 7.1
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMpotassium chlorideKCl
2.0 mMmagnesium chlorideMgCl2
1.0 mMEGTA
0.02 % (v/v)Tween20

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)
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV
DetailsActin filaments were reconstituted by adding salt to monomeric actin.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
DetailsTitan Krios G3 microscope was aligned using Sherpa (FEI). Data collected in superresolution mode.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7916 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsK3 operated in super-resolution mode.
Particle selectionNumber selected: 1569882
Startup modelType of model: EMDB MAP
EMDB ID:

15109

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Details: Refinement from Local searches in RELION. / Number images used: 1386604
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.4)
Details: First 3D refinement was performed in helical SPHIRE with meridien alpha, which imposes helical restraints to limit particle shifts to the helical rise to prevent particle duplication, but ...Details: First 3D refinement was performed in helical SPHIRE with meridien alpha, which imposes helical restraints to limit particle shifts to the helical rise to prevent particle duplication, but does not apply helical symmetry
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.1.0)
Details: 3D classification without image alignment was performed in RELION to remove particles that did not contain high-resolution information.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8a2t


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe structure was refined using phenix-real-space refine.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8oi8:
Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the R183W mutation

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