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- PDB-4mko: Crystal structure of the monomeric, cleaved form of the Pore-Form... -

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Basic information

Entry
Database: PDB / ID: 4mko
TitleCrystal structure of the monomeric, cleaved form of the Pore-Forming Toxin Monalysin
ComponentsMonalysin
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
ACETATE ION / : / Monalysin
Similarity search - Component
Biological speciesPseudomonas entomophila (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 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 5, 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 1.4Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / 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_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_alt_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.pdbx_ptnr1_label_alt_id / _struct_conn.pdbx_ptnr2_label_alt_id / _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_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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,72833
Polymers105,8334
Non-polymers2,89629
Water23,7441318
1
A: Monalysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,1087
Polymers26,4581
Non-polymers6506
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Monalysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,2269
Polymers26,4581
Non-polymers7688
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Monalysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,28510
Polymers26,4581
Non-polymers8279
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Monalysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,1087
Polymers26,4581
Non-polymers6506
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)64.844, 117.649, 150.584
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein
Monalysin


Mass: 26458.146 Da / Num. of mol.: 4 / 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: Chemical
ChemComp-HG / MERCURY (II) ION


Mass: 200.590 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Hg
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C2H3O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1318 / Source method: isolated from a natural source / Formula: H2O

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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: 2.71 Å3/Da / Density % sol: 54.67 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 0.1M sodium acetate, pH 4.6, 0.13M zinc acetate, 0.6-1.1M ammonium acetate, 2-7% PEG800, 1.5mM HgCl2, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-4 / Wavelength: 0.9393 Å
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9393 Å / Relative weight: 1
ReflectionResolution: 1.7→30 Å / Num. all: 126982 / Num. obs: 126982 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.7 % / Biso Wilson estimate: 14.11 Å2 / Rmerge(I) obs: 0.093 / Net I/σ(I): 12.9
Reflection shellResolution: 1.7→1.79 Å / Redundancy: 6.8 % / Rmerge(I) obs: 0.445 / Mean I/σ(I) obs: 3.9 / Num. unique all: 18400 / % possible all: 100

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Processing

SoftwareName: BUSTER / Version: 2.11.4 / Classification: refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PBD entry 4MJT

4mjt


Resolution: 1.7→29.78 Å / Cor.coef. Fo:Fc: 0.9404 / Cor.coef. Fo:Fc free: 0.9283 / SU R Cruickshank DPI: 0.096 / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.2046 6374 5.02 %RANDOM
Rwork0.1791 ---
obs0.1804 126896 99.82 %-
all-126982 --
Displacement parametersBiso mean: 17.56 Å2
Baniso -1Baniso -2Baniso -3
1-4.8666 Å20 Å20 Å2
2---8.8768 Å20 Å2
3---4.0102 Å2
Refine analyzeLuzzati coordinate error obs: 0.185 Å
Refinement stepCycle: LAST / Resolution: 1.7→29.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7476 0 68 1318 8862
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0097925HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.9310856HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2651SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes189HARMONIC2
X-RAY DIFFRACTIONt_gen_planes1224HARMONIC5
X-RAY DIFFRACTIONt_it7925HARMONIC20
X-RAY DIFFRACTIONt_nbd2SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion3.58
X-RAY DIFFRACTIONt_other_torsion15.34
X-RAY DIFFRACTIONt_chiral_improper_torsion1054SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies39HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact10211SEMIHARMONIC4
LS refinement shellResolution: 1.7→1.74 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.2334 500 5.38 %
Rwork0.212 8792 -
all0.2132 9292 -
obs--99.82 %

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