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- EMDB-13158: Structure of the P. aeruginosa ExoY-F-actin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-13158
TitleStructure of the P. aeruginosa ExoY-F-actin complex
Map dataLocally filtered map
Sample
  • Complex: The complex of P. aeruginosa ExoY with F-actin
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION
Function / homology
Function and homology information


calcium- and calmodulin-responsive adenylate cyclase activity / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / detection of maltose stimulus / mesenchyme migration / troponin I binding / maltose transport complex / filamentous actin / actin filament bundle ...calcium- and calmodulin-responsive adenylate cyclase activity / cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / detection of maltose stimulus / mesenchyme migration / troponin I binding / maltose transport complex / filamentous actin / actin filament bundle / maltose binding / carbohydrate transport / maltose transport / maltodextrin transmembrane transport / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / outer membrane-bounded periplasmic space / cell body / periplasmic space / hydrolase activity / protein domain specific binding / DNA damage response / calcium ion binding / positive regulation of gene expression / magnesium ion binding / extracellular region / ATP binding / identical protein binding / membrane / cytoplasm
Similarity search - Function
Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Actins signature 1. / Actin, conserved site ...Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Bacterial extracellular solute-binding protein / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Maltose/maltodextrin-binding periplasmic protein / Actin, alpha skeletal muscle / Adenylate cyclase ExoY
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / Rabbit (rabbit) / Amanita phalloides (death cap)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBelyy A / Merino F / Raunser S
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2021
Title: Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens.
Authors: Alexander Belyy / Felipe Merino / Undine Mechold / Stefan Raunser /
Abstract: Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this ...Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this activation is, however, unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus bound to their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo-state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. Other nucleotidyl cyclase toxins that bind to calmodulin rather than actin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is conserved in these enzymes, albeit the different activator.
History
DepositionJul 1, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p1g
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7p1g
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13158.png

Downloads & links

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Map

FileDownload / File: emd_13158.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.14778225 - 0.26307508
Average (Standard dev.)0.0005825963 (±0.006682268)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1480.2630.001

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Supplemental data

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Additional map: DeepEMhanced map

Fileemd_13158_additional_1.map
AnnotationDeepEMhanced map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The complex of P. aeruginosa ExoY with F-actin

EntireName: The complex of P. aeruginosa ExoY with F-actin
Components
  • Complex: The complex of P. aeruginosa ExoY with F-actin
    • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Phalloidin
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: PHOSPHATE ION

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Supramolecule #1: The complex of P. aeruginosa ExoY with F-actin

SupramoleculeName: The complex of P. aeruginosa ExoY with F-actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Macromolecule #1: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclas...

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 84.941797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE L KAKGKSAL ...String:
MGSSHHHHHH SSGLVPRGSH MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE L KAKGKSAL MFNLQEPYFT WPLIAADGGY AFKYENGKYD IKDVGVDNAG AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NK GETAMTI NGPWAWSNID TSKVNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT DEGLEAVNKD KPL GAVALK SYEEELVKDP RIAATMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDEALKDAQ TNSGSSGSSG RIDG HRQVV SNATAQPGPL LRPADMQARA LQDLFDAQGV GVPVEHALRM QAVARQTNTV FGIRPVERIV TTLIEEGFPT KGFSV KGKS SNWGPQAGFI CVDQHLSKRE DRDTAEIRKL NLAVAKGMDG GAYTQTDLRI SRQRLAELVR NFGLVADGVG PVRLLT AQG PSGKRYEFEA RQEPDGLYRI SRLGRSEAVQ VLASPACGLA MTADYDLFLV APSIEAHGSG GLDARRNTAV RYTPLGA KD PLSEDGFYGR EDMARGNITP RTRQLVDALN DCLGRGEHRE MFHHSDDAGN PGSHMGDNFP ATFYLPRAME HRVGEESV R FDEVCVVADR KSFSLLVECI KGNGYHFTAH PDWNVPLRPS FQEALDFFQR KV

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Rabbit (rabbit)
Molecular weightTheoretical: 42.096953 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #3: Phalloidin

MacromoleculeName: Phalloidin / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
W(EEP)A(DTH)C(HYP)A

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 10 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 5 / Formula: GH3
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-GH3:
3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / 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 #7: PHOSPHATE ION

MacromoleculeName: PHOSPHATE ION / type: ligand / ID: 7 / Number of copies: 5 / Formula: PO4
Molecular weightTheoretical: 94.971 Da
Chemical component information

ChemComp-PO4:
PHOSPHATE ION

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 12437 / Average exposure time: 1.5 sec. / Average electron dose: 93.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2249589
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6fhl

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 1535755
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7ad9

chain_id: B

5xnw

chain_id: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7p1g:
Structure of the P. aeruginosa ExoY-F-actin complex

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