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- PDB-8trn: Actin 1 from T. gondii in filaments bound to MgADP and jasplakinolide -

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Basic information

Entry
Database: PDB / ID: 8trn
TitleActin 1 from T. gondii in filaments bound to MgADP and jasplakinolide
ComponentsActin
KeywordsSTRUCTURAL PROTEIN / Actin / cytoskeleton / toxoplasmosis
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cytoskeleton / hydrolase activity / ATP binding / cytoplasm
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
Jasplakinolide / ADENOSINE-5'-DIPHOSPHATE / Actin
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 Å
AuthorsHvorecny, K.L. / Sladewski, T.E. / Heaslip, A.T. / Kollman, J.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F32AI145111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138316 United States
CitationJournal: Nat Commun / Year: 2024
Title: Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover.
Authors: Kelli L Hvorecny / Thomas E Sladewski / Enrique M De La Cruz / Justin M Kollman / Aoife T Heaslip /
Abstract: The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. ...The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure.
History
DepositionAug 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Actin
B: Actin
C: Actin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,35412
Polymers125,8703
Non-polymers3,4849
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Actin /


Mass: 41956.711 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Toxoplasma gondii (eukaryote) / Gene: ACT1 / Plasmid: pFastBac / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P53476, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-9UE / Jasplakinolide


Mass: 709.670 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C36H45BrN4O6 / Feature type: SUBJECT OF INVESTIGATION
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: helical reconstruction

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Sample preparation

ComponentName: Filament of Actin 1 from T. gondii with MgADP and jasplakinolide
Type: COMPLEX
Details: Actin protomers from T. gondii assemble into a two-start helix, stabilized by jasplakinolide
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Toxoplasma gondii (eukaryote) / Strain: Sf9
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFastBac
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm
Image recordingAverage exposure time: 5 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
7Coot0.9.8.1model fitting
8UCSF ChimeraX1.5model fitting
9ISOLDE1.5model fitting
11PHENIX1.2model refinement
12cryoSPARC3initial Euler assignment
13cryoSPARC4.2final Euler assignment
15PHENIX1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -167 ° / Axial rise/subunit: 28.1 Å / Axial symmetry: C1
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 2682453 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT

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