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- PDB-5ogw: Cryo-EM structure of jasplakinolide-stabilized malaria parasite F... -

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Basic information

Entry
Database: PDB / ID: 5ogw
TitleCryo-EM structure of jasplakinolide-stabilized malaria parasite F-actin at near-atomic resolution
ComponentsActin-1
KeywordsSTRUCTURAL PROTEIN / F-actin / Plasmodium / malaria parasite / cytoskeleton / cryo-EM / JAS / Jasplakinolide / filament / glideosome / gliding motility / thin filament
Function / homology
Function and homology information


actin polymerization-dependent cell-to-cell migration in host / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / hydrolase activity / ATP binding / nucleus / cytoplasm
Similarity search - Function
ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family ...ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / 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 / Nucleotidyltransferase; domain 5 / Alpha-Beta Complex / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Jasplakinolide / ADENOSINE-5'-DIPHOSPHATE / Actin-1
Similarity search - Component
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPospich, S. / Kumpula, E.-P. / von der Ecken, J. / Vahokoski, J. / Kursula, I. / Raunser, S.
Funding support Germany, Finland, 4items
OrganizationGrant numberCountry
Max Planck Society Germany
European Union615984
Academy of Finland257537, 265112, 292718 Finland
Emil Aaltonen, Sigrid Juselius, and Jane and Aatos Erkko foundations
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability.
Authors: Sabrina Pospich / Esa-Pekka Kumpula / Julian von der Ecken / Juha Vahokoski / Inari Kursula / Stefan Raunser /
Abstract: During their life cycle, apicomplexan parasites, such as the malaria parasite , use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and ...During their life cycle, apicomplexan parasites, such as the malaria parasite , use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, actin 1 (Act1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of Act1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilized Act1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.
History
DepositionJul 13, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 27, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2017Group: Data processing / Database references / Category: citation / em_software
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_software.name
Revision 1.2Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.4Nov 9, 2022Group: Database references / Refinement description / Category: database_2 / struct_ncs_oper
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Actin-1
B: Actin-1
C: Actin-1
D: Actin-1
E: Actin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,62518
Polymers210,2385
Non-polymers4,38713
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D
51E
12F
22G
32H
42I
52J

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A8 - 380
2111B8 - 380
3111C8 - 380
4111D8 - 380
5111E8 - 380
1121F1
2121G1
3121H1
4121I1
5121J1

NCS ensembles :
ID
1
2

NCS oper:
IDCodeMatrixVector
1given(1), (0.908672, -0.417502, 0.002737), (0.905021, 0.425322, -0.006226)22.44956, -13.07469
2given(-0.976351, 0.21519, -0.020781), (-0.97568, -0.218491, -0.017604), (1)90.31339, 109.74996
3given(-0.967307, -0.237444, -0.089098), (0.913366, 0.395447, -0.096869), (-0.811622, -0.582976, -0.037518)117.40153, 0.22953, 122.3214
4given(-0.956876, 0.254932, -0.139274), (-0.251032, -0.966924, -0.045185), (-0.146186, -0.008274, 0.989222)94.02023, 101.90085, 34.76189

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Components

#1: Protein
Actin-1 / / Actin I / PfACT1


Mass: 42047.676 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (isolate HB3) (eukaryote)
Strain: isolate HB3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P86287
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-9UE / Jasplakinolide


Mass: 709.670 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C36H45BrN4O6

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Plasmodium falciparum actin 1 filament stabilized by jasplakinolide
Type: COMPLEX / Details: Filament / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Plasmodium falciparum HB3 (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 10 mM HEPES pH 7.5, 0.2 mM CaCl2, 50 mM KCl, 4 mM MgCl2, 5 mM DTT and 0.5 mM ATP. JAS was added at a 1:1 molar ratio during polyermization.
Buffer component
IDConc.FormulaBuffer-ID
110 mMHEPES1
20.2 mMCaCl21
350 mMKCl1
45 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Twist (degree) 167.5 Rise (A) 27.4
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 97 % / Chamber temperature: 298 K
Details: Sample (2 uL of JAS-stabilized F-actin solution) was applied to a glow-discharged holey carbon grid, incubated for 30 s and manually blotted for 4 s from the backside with filter paper.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2700 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 110 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 1634
Image scansMovie frames/image: 24 / Used frames/image: 1-4

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Processing

SoftwareName: REFMAC / Version: 5.8.0155 / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SPARXv3.0particle selectionsxhelixboxer.py
1EPUimage acquisition
4CTFFIND4.0.7CTF correction
5RELION1.4CTF correction
8MODELLERmodel fittinghomology modelling
9UCSF Chimeramodel fittingrigid body fitting
10iMODFITmodel fittingflexible fitting
12Cootmodel refinementmanual building
13PHENIXmodel refinementinital refinement
14REFMACmodel refinementfinal refinement
15RELION1.4initial Euler assignment
16RELION1.4final Euler assignment
18RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 144058
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140716 / Actual pixel size: 1.14 Å / Symmetry type: POINT
RefinementResolution: 3.8→191.52 Å / Cor.coef. Fo:Fc: 0.843 / SU B: 37.916 / SU ML: 0.487 / ESU R: 2.247
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS RIGID BODY FITTED INTO THE ADJACENT DESNITIES CORRESPONDING TO JAS (CHAIN F, H).
RfactorNum. reflection% reflection
Rwork0.3268 --
obs0.3268 63851 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 109.718 Å2
Baniso -1Baniso -2Baniso -3
1-0.76 Å20.17 Å20.22 Å2
2--1.38 Å2-0.39 Å2
3----2.14 Å2
Refinement stepCycle: 1 / Total: 14830
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01915155
ELECTRON MICROSCOPYr_bond_other_d0.0040.0214325
ELECTRON MICROSCOPYr_angle_refined_deg1.3741.97420560
ELECTRON MICROSCOPYr_angle_other_deg1.129333105
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.64851845
ELECTRON MICROSCOPYr_dihedral_angle_2_deg23.84224.186645
ELECTRON MICROSCOPYr_dihedral_angle_3_deg8.416152595
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.3021590
ELECTRON MICROSCOPYr_chiral_restr0.0750.22270
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02117425
ELECTRON MICROSCOPYr_gen_planes_other0.0030.023290
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.52310.4617395
ELECTRON MICROSCOPYr_mcbond_other6.52310.4617394
ELECTRON MICROSCOPYr_mcangle_it10.79215.7019235
ELECTRON MICROSCOPYr_mcangle_other10.79115.7029236
ELECTRON MICROSCOPYr_scbond_it7.93211.587760
ELECTRON MICROSCOPYr_scbond_other7.93211.587761
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other13.416.97511326
ELECTRON MICROSCOPYr_long_range_B_refined20.05345725
ELECTRON MICROSCOPYr_long_range_B_other20.05345726
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.5

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A57408.72
11B57408.7
11C574012.37
11D57408.21
11E57407.26
22A9112.94
22B9110.94
22C9112.68
22D9132.37
22E917.79
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.564 4675 -
Rfree-0 -
obs--100 %

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