EMDB-16889: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring ...

Basic information

Entry

Database: EMDB / ID: EMD-16889

• Title: Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the N111S mutation
• Map data: Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the N111S mutation.

Sample

• Complex: Actin filament harboring the N111S mutation.
  • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: water

• Keywords: Actin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN

Function / homology

Function:

positive regulation of norepinephrine uptake / cellular response to cytochalasin B / Formation of the embryonic stem cell BAF (esBAF) complex / regulation of transepithelial transport / Formation of the canonical BAF (cBAF) complex / morphogenesis of a polarized epithelium / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / Formation of the polybromo-BAF (pBAF) complex / structural constituent of postsynaptic actin cytoskeleton / Gap junction degradation / GBP-mediated host defense / Formation of neuronal progenitor and neuronal BAF (npBAF and nBAF) / protein localization to adherens junction / Formation of the non-canonical BAF (ncBAF) complex / Cell-extracellular matrix interactions / regulation of G0 to G1 transition / dense body / Tat protein binding / Folding of actin by CCT/TriC / postsynaptic actin cytoskeleton / Regulation of CDH1 Function / apical protein localization / Adherens junctions interactions / microtubule nucleation / Prefoldin mediated transfer of substrate to CCT/TriC / RHOF GTPase cycle / adherens junction assembly / regulation of double-strand break repair / Sensory processing of sound by outer hair cells of the cochlea / tight junction / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / positive regulation of T cell differentiation / maintenance of blood-brain barrier / Interaction between L1 and Ankyrins / apical junction complex / regulation of nucleotide-excision repair / positive regulation of stem cell population maintenance / NuA4 histone acetyltransferase complex / regulation of norepinephrine uptake / transporter regulator activity / Recycling pathway of L1 / positive regulation of double-strand break repair / cortical cytoskeleton / Regulation of MITF-M-dependent genes involved in pigmentation / negative regulation of cell differentiation / establishment or maintenance of cell polarity / nitric-oxide synthase binding / brush border / EPH-ephrin mediated repulsion of cells / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / kinesin binding / regulation of protein localization to plasma membrane / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of G1/S transition of mitotic cell cycle / axonogenesis / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / calyx of Held / cell motility / nitric-oxide synthase regulator activity / FCGR3A-mediated phagocytosis / actin filament / Translocation of SLC2A4 (GLUT4) to the plasma membrane / adherens junction / positive regulation of cell differentiation / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / VEGFA-VEGFR2 Pathway / B-WICH complex positively regulates rRNA expression / tau protein binding / Schaffer collateral - CA1 synapse / DNA Damage Recognition in GG-NER / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / kinetochore / nuclear matrix / Signaling by RAF1 mutants / cell-cell junction / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / actin cytoskeleton / Signaling by BRAF and RAF1 fusions / UCH proteinases / lamellipodium / presynapse / HATs acetylate histones / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production

Domain/homology:

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

Component:

Actin, cytoplasmic 1

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 2.3 Å
• Authors: Oosterheert W / Blanc FEC / Roy A / Belyy A / Hofnagel O / Hummer G / Bieling P / Raunser S

Funding support

Germany, European Union, 3 items

Organization	Grant number	Country
Max Planck Society		Germany
Alexander von Humboldt Foundation		Germany
European Research Council (ERC)	856118	European Union

• Citation: Journal: 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 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

Deposition	Mar 22, 2023	-
Header (metadata) release	Aug 9, 2023	-
Map release	Aug 9, 2023	-
Update	Nov 22, 2023	-
Current status	Nov 22, 2023	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_16889.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the N111S mutation.
• Voxel size: X=Y=Z: 0.695 Å

Density

Contour Level	By AUTHOR: 0.0375
Minimum - Maximum	-0.16569497 - 0.39300117
Average (Standard dev.)	0.00015015746 (±0.0064122896)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 384 384 384 Spacing 384 384 384
Cell	A=B=C: 266.88 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_16889_msk_1.map

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

• File: emd_16889_additional_1.map
• Annotation: 3D refined, unsharpened cryo-EM density map of filamentous beta-actin harboring the N111S mutation.

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

• File: emd_16889_half_map_1.map
• Annotation: Half map 1 of the refinement of filamentous beta-actin harboring the N111S mutation.

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

• File: emd_16889_half_map_2.map
• Annotation: Half map 2 of the refinement of filamentous beta-actin harboring the N111S mutation.

Sample components

Entire : Actin filament harboring the N111S mutation.

• Entire: Name: Actin filament harboring the N111S mutation.

Components

• Complex: Actin filament harboring the N111S mutation.
  • Protein or peptide: Actin, cytoplasmic 1, N-terminally processed
• Ligand: ADENOSINE-5'-DIPHOSPHATE
• Ligand: MAGNESIUM ION
• Ligand: water

Supramolecule #1: Actin filament harboring the N111S mutation.

• Supramolecule: Name: Actin filament harboring the N111S mutation. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1; 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.
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Actin, cytoplasmic 1, N-terminally processed

• Macromolecule: Name: Actin, cytoplasmic 1, N-terminally processed / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 41.73659 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

String:

MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LSPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESAGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

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

Macromolecule #3: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #4: water

• Macromolecule: Name: water / type: ligand / ID: 4 / Number of copies: 616 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: filament

Sample preparation

• Concentration: 2.5 mg/mL

Buffer

pH: 7.1
Component:

Concentration	Name	Formula
10.0 mM	HEPES
100.0 mM	potassium chloride	KCl
2.0 mM	magnesium chloride	MgCl2
1.0 mM	EGTA
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)

• Grid: Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
• Vitrification: Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV
• Details: Actin filaments were reconstituted by adding salt to monomeric actin.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
• Details: Titan Krios G3 microscope was aligned using Sherpa (FEI). Data collected in superresolution mode.
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 9516 / Average electron dose: 70.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 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 stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: K3 operated in super-resolution mode.
• Particle selection: Number selected: 2001281 / Details: crYOLO in filament mode.

Startup model

Type of model: EMDB MAP
EMDB ID:

15109

• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0); Details: The final refinement was performed from local searches in RELION.; Number images used: 1756928
• Initial angle assignment: Type: 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 does not apply helical symmetry
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
• Final 3D classification: Number 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.

Atomic model buiding 1

Initial model

PDB ID:

8a2t

Chain - Source name: PDB / Chain - Initial model type: experimental model

• 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.
• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Output model

PDB-8oid:
Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the N111S mutation