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- EMDB-13865: Leishmania major ADP-actin filament decorated with Leishmania maj... -

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Basic information

Entry
Database: EMDB / ID: EMD-13865
TitleLeishmania major ADP-actin filament decorated with Leishmania major cofilin
Map dataAutosharpened and real-space symmetrized map from postprocess
Sample
  • Complex: ADP-state actin filament decorated with Leishmania major cofilin.
    • Protein or peptide: Actin
    • Protein or peptide: ADF/Cofilin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin / Filament / Parasite / STRUCTURAL PROTEIN / cofilin / ADP / decorated
Function / homology
Function and homology information


kinetoplast / nuclear lumen / actin filament depolymerization / ciliary plasm / intracellular transport / actin filament binding / actin cytoskeleton / endonuclease activity / cytoskeleton / chromatin remodeling ...kinetoplast / nuclear lumen / actin filament depolymerization / ciliary plasm / intracellular transport / actin filament binding / actin cytoskeleton / endonuclease activity / cytoskeleton / chromatin remodeling / cell division / ATP binding / cytoplasm
Similarity search - Function
ADF/Cofilin / Actin-depolymerising factor homology domain / Cofilin/tropomyosin-type actin-binding protein / ADF-H domain profile. / Actin depolymerisation factor/cofilin -like domains / ADF-H/Gelsolin-like domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site ...ADF/Cofilin / Actin-depolymerising factor homology domain / Cofilin/tropomyosin-type actin-binding protein / ADF-H domain profile. / Actin depolymerisation factor/cofilin -like domains / ADF-H/Gelsolin-like domain superfamily / 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: 3.4 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_13865.png

Downloads & links

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Map

FileDownload / File: emd_13865.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-10.242599999999999 - 28.928706999999999
Average (Standard dev.)0.000000000004647 (±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_13865_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocess map from Relion

Fileemd_13865_additional_1.map
AnnotationPostprocess map from Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_13865_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_13865_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ADP-state actin filament decorated with Leishmania major cofilin.

EntireName: ADP-state actin filament decorated with Leishmania major cofilin.
Components
  • Complex: ADP-state actin filament decorated with Leishmania major cofilin.
    • Protein or peptide: Actin
    • Protein or peptide: ADF/Cofilin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ADP-state actin filament decorated with Leishmania major cofilin.

SupramoleculeName: ADP-state actin filament decorated with Leishmania major cofilin.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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: ADF/Cofilin

MacromoleculeName: ADF/Cofilin / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Leishmania major (eukaryote)
Molecular weightTheoretical: 15.606783 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
AISGVTLEES VRGAIDDLRM KKSRYVMMCI GADGKKIEVT EVGERGVNYT DLKEKFSAEK PCYVAFDFEY NDAGSKREKL ILIQWIPDT ARPREKMMYS ASRDALSSVS EGYLPIQAND ESGLDAEEII RKVRLHRSV

UniProtKB: ADF/Cofilin

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / 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 #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

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.
Details3 ul of 12 uM Leishmania actin was applied on the grid and left to settle for 15 s at room temperature. This grid was then mounted to the vitrobot and 1 ul of actin was withdrawn. On top of the remaining 2 ul of Leishmania actin, 1 ul of Leishmania cofilin at 77 uM

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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: 28.56 Å
Applied symmetry - Helical parameters - Δ&Phi: -161.26 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 46929
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-7q8s:
Leishmania major ADP-actin filament decorated with Leishmania major cofilin

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