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

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Basic information

Entry
Database: PDB / ID: 5cxl
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- and calmodulin-responsive adenylate cyclase activity / hemolysis in another organism / adenylate cyclase / cAMP biosynthetic process / adenylate cyclase activity / channel activity / toxin activity / positive regulation of cytosolic calcium ion concentration / calmodulin binding / calcium ion binding ...calcium- and calmodulin-responsive adenylate cyclase activity / hemolysis in another organism / adenylate cyclase / cAMP biosynthetic process / adenylate cyclase activity / channel activity / toxin activity / positive regulation of cytosolic calcium ion concentration / 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 / Haemolysin-type calcium binding-related / Haemolysin-type calcium binding protein related domain / RTX toxin determinant A / 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 ...RTX, pore-forming domain / N-terminal domain in RTX protein / Haemolysin-type calcium binding-related / Haemolysin-type calcium binding protein related domain / RTX toxin determinant A / 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
NITRATE ION / Bifunctional hemolysin/adenylate cyclase
Similarity search - Component
Biological speciesBordetella pertussis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.45 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 29, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Sep 2, 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 / diffrn_radiation_wavelength / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional hemolysin/adenylate cyclase
B: Bifunctional hemolysin/adenylate cyclase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,81320
Polymers32,0482
Non-polymers76518
Water3,981221
1
A: Bifunctional hemolysin/adenylate cyclase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,40710
Polymers16,0241
Non-polymers3839
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Bifunctional hemolysin/adenylate cyclase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,40710
Polymers16,0241
Non-polymers3839
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)52.225, 52.225, 195.952
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-1825-

HOH

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Components

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


Mass: 16024.102 Da / Num. of mol.: 2 / Fragment: BLOCK V OF RTX DOMAIN (UNP RESISDUES 1529-1681)
Source method: isolated from a genetically manipulated source
Details: THE LAST 5 AMINO ACIDS ARE TOO FLEXIBLE TO BE FIT IN THE ELECTRON DENSITY MAP.
Source: (gene. exp.) Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) (bacteria)
Strain: Tohama I / ATCC BAA-589 / NCTC 13251 / Gene: cya, cyaA, BP0760 / Plasmid: PET42B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DKX7, adenylate cyclase
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: NO3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 221 / 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: 2.08 Å3/Da / Density % sol: 40.99 % / Description: COLOURLESS, CUBE (a=100 um)
Crystal growTemperature: 291.15 K / Method: vapor diffusion, sitting 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 NITRATE, 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: 0.91841 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: 0.91841 Å / Relative weight: 1
ReflectionResolution: 1.45→50 Å / Num. obs: 46709 / % possible obs: 99.9 % / Observed criterion σ(I): -3 / Redundancy: 8.55 % / Rmerge(I) obs: 0.056 / Net I/σ(I): 22.11
Reflection shellResolution: 1.45→1.54 Å / Rmerge(I) obs: 0.787 / Mean I/σ(I) obs: 2.55 / % possible all: 99.8

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Processing

Software
NameVersionClassification
MOSFLMdata reduction
PHASERphasing
REFMAC5.7.0029refinement
XDSdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 5CVW

5cvw


Resolution: 1.45→32.15 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.964 / SU B: 2.655 / SU ML: 0.046 / Cross valid method: THROUGHOUT / ESU R: 0.072 / ESU R Free: 0.072 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: USED WEIGHTING FACTOR 2.5 AND ANISOTROPIC THERMAL FACTORS WERE REFINED
RfactorNum. reflection% reflectionSelection details
Rfree0.207 2458 5 %RANDOM
Rwork0.148 ---
obs0.151 46709 99.9 %-
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: 22.57 Å2
Baniso -1Baniso -2Baniso -3
1--0 Å20 Å2-0 Å2
2---0 Å20 Å2
3---0.01 Å2
Refinement stepCycle: LAST / Resolution: 1.45→32.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2172 0 24 221 2417
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0190.0192397
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.921.933280
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1635336
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.03725.373134
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.33115354
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.1281516
X-RAY DIFFRACTIONr_chiral_restr0.1480.2347
X-RAY DIFFRACTIONr_gen_planes_refined0.010.021982
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_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr6.1832397
X-RAY DIFFRACTIONr_sphericity_free29.623583
X-RAY DIFFRACTIONr_sphericity_bonded19.23952499
LS refinement shellResolution: 1.45→1.49 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.275 176 -
Rwork0.185 3344 -
obs--99.72 %

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