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- EMDB-13864: Leishmania major actin filament in ADP-state -

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Basic information

Entry
Database: EMDB / ID: EMD-13864
TitleLeishmania major actin filament in ADP-state
Map dataAutosharpened and real-space symmetrized map from postprocess
Sample
  • Complex: Bare, undecorated, ADP-state actin filament from the sample mixed with Leishmania major cofilin.
    • Protein or peptide: Actin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsActin / Filament / Parasite / ADP-Pi / STRUCTURAL PROTEIN
Function / homology
Function and homology information


kinetoplast / actin cytoskeleton / endonuclease activity / chromatin remodeling
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
Biological speciesLeishmania major (eukaryote)
Methodhelical reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsKotila T / Muniyandi S / Lappalainen P / Huiskonen JT
Funding support Finland, 2 items
OrganizationGrant numberCountry
Academy of Finland320161 Finland
Jane and Aatos Erkko Foundation4708679 Finland
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite.
Authors: Tommi Kotila / Hugo Wioland / Muniyandi Selvaraj / Konstantin Kogan / Lina Antenucci / Antoine Jégou / Juha T Huiskonen / Guillaume Romet-Lemonne / Pekka Lappalainen /
Abstract: Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, ...Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin 'cofactors', which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.
History
DepositionNov 11, 2021-
Header (metadata) releaseMay 18, 2022-
Map releaseMay 18, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13864.png

Downloads & links

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Map

FileDownload / File: emd_13864.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAutosharpened and real-space symmetrized map from postprocess
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 288 pix.
= 248.17 Å		0.86 Å/pix.
x 288 pix.
= 248.17 Å		0.86 Å/pix.
x 288 pix.
= 248.17 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8617 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-11.464039 - 34.764954000000003
Average (Standard dev.)-0.000000000002834 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 248.1696 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13864_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: #1

Fileemd_13864_additional_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: Half-map2

Fileemd_13864_half_map_1.map
AnnotationHalf-map2
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half-map1

Fileemd_13864_half_map_2.map
AnnotationHalf-map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bare, undecorated, ADP-state actin filament from the sample mixed...

EntireName: Bare, undecorated, ADP-state actin filament from the sample mixed with Leishmania major cofilin.
Components
  • Complex: Bare, undecorated, ADP-state actin filament from the sample mixed with Leishmania major cofilin.
    • Protein or peptide: Actin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Bare, undecorated, ADP-state actin filament from the sample mixed...

SupramoleculeName: Bare, undecorated, ADP-state actin filament from the sample mixed with Leishmania major cofilin.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Leishmania major (eukaryote)

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Macromolecule #1: Actin

MacromoleculeName: Actin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Leishmania major (eukaryote)
Molecular weightTheoretical: 42.063867 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MADNEQSSIV CDNGSGMVKA GFSGDDAPRH VFPSIVGRPK NMQAMMGSAN KTVYVGDEAQ SKRGVLSLKY PIEHGIVTNW DDMEKIWHH TFYNELRVNP EQHNVLLTEA PMNPKQNREK MTQIMFETFN VPSLYIGIQA VLSLYSSGRT TGIVLDAGDG V THTVPIYE ...String:
MADNEQSSIV CDNGSGMVKA GFSGDDAPRH VFPSIVGRPK NMQAMMGSAN KTVYVGDEAQ SKRGVLSLKY PIEHGIVTNW DDMEKIWHH TFYNELRVNP EQHNVLLTEA PMNPKQNREK MTQIMFETFN VPSLYIGIQA VLSLYSSGRT TGIVLDAGDG V THTVPIYE GYSLPHAVRR VDMAGRDLTE YLMKIMMETG TTFTTTAEKE IVRNVKEQLC YVALDFEEEM TNSAKSANEE AF ELPDGNV MMVGNQRFRC PEVLFKPSLI GLDEAPGFPE MVYQSINKCD IDVRRELYGN IVLSGGSTMF LNLPERLAKE ISN LAPSSI KPKVVAPPER KYSVWIGGSI LSSLTTFQTM WVKKSEYDES GPSIVHNKCF

UniProtKB: Actin

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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: 360 / 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
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Details: 10 mM HEPES, 125 mM NaCl, 5 mM KCl, 0.2 mM ATP, 0.4 mM EGTA, 1 mM MgCl2, 1 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 279.15 K / Instrument: FEI VITROBOT MARK IV
Details: blot for 5 seconds before plunging, blot force 15.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 50 / Average electron dose: 55.0 e/Å2
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: 28.4 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.54 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 232527
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6djo


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-7q8c:
Leishmania major actin filament in ADP-state

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