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- PDB-5cvw: CRYSTAL STRUCTURE OF RTX DOMAIN BLOCK V OF ADENYLATE CYCLASE TOXI... -

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Basic information

Entry
Database: PDB / ID: 5cvw
TitleCRYSTAL STRUCTURE OF RTX DOMAIN BLOCK V OF ADENYLATE CYCLASE TOXIN FROM BORDETELLA PERTUSSIS
ComponentsBifunctional hemolysin/adenylate cyclase
KeywordsTOXIN / ADENYLATE CYCLASE / RTX MOTIFS / CALCIUM BINDING
Function / homology
Function and homology information


calcium ion sequestering activity / calcium- and calmodulin-responsive adenylate cyclase activity / hemolysis in another organism / adenylate cyclase / cAMP biosynthetic process / channel activity / toxin activity / calmodulin binding / calcium ion binding / host cell plasma membrane ...calcium ion sequestering activity / calcium- and calmodulin-responsive adenylate cyclase activity / hemolysis in another organism / adenylate cyclase / cAMP biosynthetic process / channel activity / toxin activity / calmodulin binding / calcium ion binding / host cell plasma membrane / extracellular region / ATP binding / membrane
Similarity search - Function
RTX, pore-forming domain / N-terminal domain in RTX protein / RTX toxin determinant A / Haemolysin-type calcium binding-related / Haemolysin-type calcium binding protein related domain / : / Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit ...RTX, pore-forming domain / N-terminal domain in RTX protein / RTX toxin determinant A / Haemolysin-type calcium binding-related / Haemolysin-type calcium binding protein related domain / : / Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / Hemolysin-type calcium-binding conserved site / Hemolysin-type calcium-binding region signature. / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal
Similarity search - Domain/homology
Bifunctional hemolysin/adenylate cyclase
Similarity search - Component
Biological speciesBordetella pertussis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.25 Å
AuthorsMotlova, L. / Barinka, C. / Bumba, L.
Funding support Czech Republic, 2items
OrganizationGrant numberCountry
Grant Agency of the Czech RepublicGA15-11851S Czech Republic
Charles University Grant Agency353388 Czech Republic
CitationJournal: Mol Cell / Year: 2016
Title: Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.
Authors: Ladislav Bumba / Jiri Masin / Pavel Macek / Tomas Wald / Lucia Motlova / Ilona Bibova / Nela Klimova / Lucie Bednarova / Vaclav Veverka / Michael Kachala / Dmitri I Svergun / Cyril Barinka / Peter Sebo /
Abstract: Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, ...Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.
History
DepositionJul 27, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Sep 9, 2015Provider: repository / Type: Initial release
Revision 1.1Apr 20, 2016Group: Database references
Revision 1.2Jan 10, 2024Group: 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 / pdbx_struct_special_symmetry / struct_conn
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_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.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.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_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_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional hemolysin/adenylate cyclase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,73416
Polymers16,0241
Non-polymers71015
Water2,234124
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area950 Å2
ΔGint-20 kcal/mol
Surface area7900 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.824, 62.983, 77.627
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number23
Space group name H-MI222
Components on special symmetry positions
IDModelComponents
11A-1714-

MG

21A-1848-

HOH

31A-1854-

HOH

41A-1877-

HOH

51A-1899-

HOH

61A-1918-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Bifunctional hemolysin/adenylate cyclase / AC-HLY / ACT / Cyclolysin


Mass: 16024.102 Da / Num. of mol.: 1 / Fragment: BLOCK V OF RTX DOMAIN (UNP RESIDUES 1529-1681)
Source method: isolated from a genetically manipulated source
Details: The last 3 amino acids are too flexible to be fit in the electron density map.
Source: (gene. exp.) Bordetella pertussis (strain ATCC 9797 / DSM 5571 / NCTC 10739 / 18323) (bacteria)
Gene: cya, cyaA, BN118_0468 / Plasmid: PET42B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: J7QLC0, adenylate cyclase

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Non-polymers , 5 types, 139 molecules

#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 124 / 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: 2.4 Å3/Da / Density % sol: 48.67 % / Description: colourless, cube (a=90 um)
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Buffer composition: 5 mM Tris-HCl pH=7.4, 150 mM NaCl, 10 mM CaCl2 Precipitant composition: 0.2 M Magnesium sulfate, 20% v/v PEG 3350
PH range: 7

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Data collection

DiffractionMean temperature: 90 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.918 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jul 25, 2012 / Details: 2 MIRRORS
RadiationMonochromator: Si-111 CRYSTAL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918 Å / Relative weight: 1
ReflectionResolution: 1.23→50 Å / Num. obs: 42131 / % possible obs: 93.6 % / Observed criterion σ(I): -3 / Redundancy: 4.48 % / Rmerge(I) obs: 0.043 / Net I/σ(I): 19.78
Reflection shellResolution: 1.23→1.3 Å / % possible all: 72.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0123refinement
MOSFLMdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1SRP

1srp


Resolution: 1.25→29.24 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.961 / SU B: 1.507 / SU ML: 0.029 / Cross valid method: THROUGHOUT / ESU R: 0.041 / ESU R Free: 0.04 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.16644 2083 5.1 %RANDOM
Rwork0.13868 ---
obs0.14009 39003 95.71 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 17.732 Å2
Baniso -1Baniso -2Baniso -3
1-0.17 Å20 Å20 Å2
2--0.22 Å20 Å2
3----0.39 Å2
Refinement stepCycle: LAST / Resolution: 1.25→29.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1107 0 32 124 1263
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0480.0191339
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg2.2031.9491731
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2765175
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.95425.14768
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.86415194
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.681159
X-RAY DIFFRACTIONr_chiral_restr0.1630.2184
X-RAY DIFFRACTIONr_gen_planes_refined0.0130.021028
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.6881.305696
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it2.2191.993848
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it2.9061.639642
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined4.11513.031948
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr5.06631338
X-RAY DIFFRACTIONr_sphericity_free37.188535
X-RAY DIFFRACTIONr_sphericity_bonded19.90451351
LS refinement shellResolution: 1.25→1.282 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.202 120 -
Rwork0.171 2201 -
obs--73.5 %
Refinement TLS params.Method: refined / Origin x: 15.5006 Å / Origin y: -7.0153 Å / Origin z: 11.8473 Å
111213212223313233
T0.002 Å2-0.0009 Å2-0.0009 Å2-0.0014 Å20.0012 Å2--0.0254 Å2
L0.098 °20.0018 °2-0.0406 °2-0.0916 °20.0087 °2--0.0361 °2
S-0.0014 Å °-0.0087 Å °-0.0038 Å °0.0051 Å °0 Å °0.0007 Å °0.0067 Å °0.0002 Å °0.0014 Å °

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