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- PDB-4mjt: Crystal structure of the oligomeric pore-forming toxin pro-Monalysin -

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Basic information

Entry
Database: PDB / ID: 4mjt
TitleCrystal structure of the oligomeric pore-forming toxin pro-Monalysin
Components
  • MONALYSIN
  • Monalysin
KeywordsTOXIN / Pore-Forming Toxin
Function / homology
Function and homology information


hemolysis in another organism / porin activity / pore complex / monoatomic ion transport / protein homooligomerization / toxin activity / host cell plasma membrane / extracellular region
Similarity search - Function
Monalysin, beta barrel Pore-forming domain / Beta barrel Pore-forming domain
Similarity search - Domain/homology
Biological speciesPseudomonas entomophila (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.85 Å
AuthorsLeone, P. / Roussel, A.
CitationJournal: J Biol Chem / Year: 2015
Title: X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.
Authors: Philippe Leone / Cecilia Bebeacua / Onya Opota / Christine Kellenberger / Bruno Klaholz / Igor Orlov / Christian Cambillau / Bruno Lemaitre / Alain Roussel /
Abstract: β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage ...β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.
History
DepositionSep 4, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2015Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2015Group: Database references
Revision 1.2Apr 22, 2015Group: Database references
Revision 1.3Jun 10, 2015Group: Database references
Revision 1.4Feb 28, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.content_type / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: MONALYSIN
B: MONALYSIN
C: MONALYSIN
D: MONALYSIN
E: MONALYSIN
F: MONALYSIN
G: MONALYSIN
H: MONALYSIN
I: MONALYSIN
J: Monalysin
K: Monalysin
L: Monalysin
M: Monalysin
N: Monalysin
O: Monalysin
P: Monalysin
Q: Monalysin
R: Monalysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,86445
Polymers264,09818
Non-polymers1,76627
Water11,818656
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31420 Å2
ΔGint-1009 kcal/mol
Surface area80210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)162.400, 146.200, 144.320
Angle α, β, γ (deg.)90.00, 122.80, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
MONALYSIN


Mass: 26458.146 Da / Num. of mol.: 9 / Fragment: UNP residues 36-271
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas entomophila (bacteria) / Strain: L48 / Gene: PSEEN3174 / Production host: Escherichia coli (E. coli) / References: UniProt: Q1I8U1
#2: Protein/peptide
Monalysin


Mass: 2886.110 Da / Num. of mol.: 9 / Fragment: UNP residues 9-35
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas entomophila (bacteria) / Strain: L48 / Gene: PSEEN3174 / Production host: Escherichia coli (E. coli) / References: UniProt: Q1I8U1
#3: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 27 / Source method: obtained synthetically / Formula: Zn
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 656 / Source method: isolated from a natural source / Formula: H2O
Compound detailsAUTHORS STATE THAT PEPTIDE CHAIN (RESIDUES 9-35) AND PROTEIN CHAIN (RESIDUES 36-271) BELONG TO A ...AUTHORS STATE THAT PEPTIDE CHAIN (RESIDUES 9-35) AND PROTEIN CHAIN (RESIDUES 36-271) BELONG TO A SAME SINGLE CHAIN WITHOUT BEEN CLEAVED BETWEEN RESIDUES 35 AND 36. AUTHORS HAVE STRONG EVIDENCE OF THE DOMAIN SWAPPING BUT NO EXPERIMENTAL DATA COULD PERMIT TO ASSIGN THE CHAINS UNAMBIGUOUSLY. THEREFORE, THEY WERE ASSIGNED DISTINCT CHAIN IDS.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.73 Å3/Da / Density % sol: 54.89 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 0.1M sodium acetate, 0.13M zinc acetate, 0.6-1.1M ammonium acetate, 2-7% PEG8000, pH 4.6, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.8726 Å
RadiationScattering type: x-ray
Radiation wavelengthWavelength: 0.8726 Å / Relative weight: 1
ReflectionResolution: 2.85→30 Å / Num. all: 65957 / Num. obs: 65957 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.4 % / Biso Wilson estimate: 73.63 Å2 / Rmerge(I) obs: 0.095 / Net I/σ(I): 13
Reflection shellResolution: 2.85→3 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.657 / Mean I/σ(I) obs: 2.1 / Num. unique all: 9623 / % possible all: 100

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
SHARPphasing
BUSTER2.11.4refinement
XDSdata reduction
SCALAdata scaling
RefinementMethod to determine structure: SAD / Resolution: 2.85→29.34 Å / Cor.coef. Fo:Fc: 0.9311 / Cor.coef. Fo:Fc free: 0.9072 / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.2162 3346 5.07 %RANDOM
Rwork0.1821 ---
obs0.1838 65948 99.84 %-
all-65957 --
Displacement parametersBiso mean: 55.54 Å2
Baniso -1Baniso -2Baniso -3
1-5.3871 Å20 Å2-3.1246 Å2
2---3.9008 Å20 Å2
3----1.4863 Å2
Refine analyzeLuzzati coordinate error obs: 0.307 Å
Refinement stepCycle: LAST / Resolution: 2.85→29.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17469 0 27 656 18152
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.00817928HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.9924436HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d5860SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes430HARMONIC2
X-RAY DIFFRACTIONt_gen_planes2626HARMONIC5
X-RAY DIFFRACTIONt_it17928HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion2.73
X-RAY DIFFRACTIONt_other_torsion18.76
X-RAY DIFFRACTIONt_chiral_improper_torsion2374SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact19705SEMIHARMONIC4
LS refinement shellResolution: 2.85→2.92 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.2731 241 4.97 %
Rwork0.2194 4612 -
all0.222 4853 -
obs--99.84 %

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