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- EMDB-21097: Negative stain electron microscopy of PmPV2, a toxin member of th... -

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Basic information

Entry
Database: EMDB / ID: EMD-21097
TitleNegative stain electron microscopy of PmPV2, a toxin member of the Membrane Attack Complex-Perforin (MACPF) from the eggs of Pomacea maculata
Map dataPmPV2
Sample
  • Complex: Perivitellin PmPV2
    • Protein or peptide: Tachylectin subunit (PmPV2-31)
    • Protein or peptide: MACPF subunit (PmPV2-67)
KeywordsPerivitellin-2 / Pomacea maculata / PmPV2 / Apple snail / Gastropod reproduction / Anti-predator defense. / TOXIN
Biological speciesPomacea maculata (invertebrata)
Methodsingle particle reconstruction / negative staining / Resolution: 15.2 Å
AuthorsOtero LH / Brola TR / Giglio ML / Ituarte S / Dreon MS / Heras H
Funding support Argentina, 3 items
OrganizationGrant numberCountry
National Research Council (Argentina)PICT 2014-0850 Argentina
National Research Council (Argentina)PICT 2015-0621 Argentina
National Research Council (Argentina)PICT-2016-1425 Argentina
CitationJournal: J Struct Biol / Year: 2020
Title: Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.
Authors: M L Giglio / S Ituarte / V Milesi / M S Dreon / T R Brola / J Caramelo / J C H Ip / S Maté / J W Qiu / L H Otero / H Heras /
Abstract: The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are ...The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.
History
DepositionDec 10, 2019-
Header (metadata) releaseDec 25, 2019-
Map releaseOct 21, 2020-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21097.png

Downloads & links

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Map

FileDownload / File: emd_21097.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPmPV2
Voxel sizeX=Y=Z: 2.02 Å
Density
Contour LevelBy AUTHOR: 4.6 / Movie #1: 4.6
Minimum - Maximum-4.9147325 - 13.074539
Average (Standard dev.)-0.015995676 (±0.62559587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 387.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.022.022.02
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z387.840387.840387.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-4.91513.075-0.016

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Supplemental data

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Sample components

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Entire : Perivitellin PmPV2

EntireName: Perivitellin PmPV2
Components
  • Complex: Perivitellin PmPV2
    • Protein or peptide: Tachylectin subunit (PmPV2-31)
    • Protein or peptide: MACPF subunit (PmPV2-67)

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Supramolecule #1: Perivitellin PmPV2

SupramoleculeName: Perivitellin PmPV2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Protein from the Pomacea maculata eggs. Its structure consists of a dimer of heterodimer, each consisting in a Tachylectin (PmPV2-31) and a MACPF-containing protein (PmPV2-67) held together by a disulfide bond.
Source (natural)Organism: Pomacea maculata (invertebrata)
Molecular weightTheoretical: 162 KDa

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Macromolecule #1: Tachylectin subunit (PmPV2-31)

MacromoleculeName: Tachylectin subunit (PmPV2-31) / type: protein_or_peptide / ID: 1 / Details: Protein without signal peptide / Enantiomer: LEVO
Source (natural)Organism: Pomacea maculata (invertebrata)
SequenceString: FTSVKLPRDE HWPYNYVSVG PAGVWAVNRQ NKLFYRTGTY GDNANMGSGW QFKQDGVGQV DVGKDKVGY INLSGGSLFR IEGISQANPV GGTPKSWEWW TKYIGMSLRE DTRFSSRIEN Q NKVLTFTF RTCFWASRIT NWCFADSSYT ETVTAGGSGT WITKSQLKYK ...String:
FTSVKLPRDE HWPYNYVSVG PAGVWAVNRQ NKLFYRTGTY GDNANMGSGW QFKQDGVGQV DVGKDKVGY INLSGGSLFR IEGISQANPV GGTPKSWEWW TKYIGMSLRE DTRFSSRIEN Q NKVLTFTF RTCFWASRIT NWCFADSSYT ETVTAGGSGT WITKSQLKYK SGTFGNPDTE GG DWILVDS GSFQHVSSGS GVVLAVRSNG ELVQRTGITC SLPQGTGWTS MLNSMSRVDT YGT VAWAVD TAGDLYFINL

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Macromolecule #2: MACPF subunit (PmPV2-67)

MacromoleculeName: MACPF subunit (PmPV2-67) / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Pomacea maculata (invertebrata)
SequenceString: ARVCPKIVPG LDKLRVGVDI TKLDLLPLFD LGDNGFRSAV ADYTCDRGQT AVVDGESFDV PDQVDSVVI ESSGQQTSSV TTIKSESQIS QALSISAGIS VETAKAGFSS SASYAEMQEA I TKYGRTVS QMSAVYTTCS ANLSPNLLLG QNPLQTLSRL PSDFTADTQG ...String:
ARVCPKIVPG LDKLRVGVDI TKLDLLPLFD LGDNGFRSAV ADYTCDRGQT AVVDGESFDV PDQVDSVVI ESSGQQTSSV TTIKSESQIS QALSISAGIS VETAKAGFSS SASYAEMQEA I TKYGRTVS QMSAVYTTCS ANLSPNLLLG QNPLQTLSRL PSDFTADTQG YYDFIKTYGT HY FNKGKLG GMFLFTSETD MSYFQNKNSQ QIEATVKATF ASILSTETGG SSDESKEVIE FKE SSLITS KFFGGQTNLA ADGLTKWQPT IAKLPYFMSG TLSTISSLIA DTTKRASMEL AVKN YLLKA KVANLDRLTY IRLNSWSVGH NELRDLSAQL QNLKTKTIFS DADEKLLQSI EDQVS VPAW FSDRTTFCFR STAVGSADQC NGQSTNTLCA EPNRYTQQYM DKTYLGDTGC RLVWKI STT ESTDWFKSVK VNFRWYPTWS PCACGPVGTP FTISAPANSW TQDYLDVTNP KFGECML QW MIEVPPTATL WAKNLEFCID FTCGKKKQCV DANQWTEPYL DISAHEACGM SWALIAK

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8.5 / Component:
ConcentrationFormula
20.0 mMTris-HCl
150.0 mMNaCl
StainingType: NEGATIVE / Material: Uranyl Acetate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.037 kPa / Details: Negative glow discharge 25 mA
DetailsThe sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 60 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2 / Details: About 400 particles per image
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 73000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 20115
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 19 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 15.2 Å / Resolution method: FSC 0.5 CUT-OFF / Number images used: 15317
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cisTEM (ver. 1.0.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cisTEM (ver. 1.0.0)
Final 3D classificationNumber classes: 19 / Software - Name: cisTEM (ver. 1.0.0)

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