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- PDB-6yhk: Crystal structure of full-length CNFy (C866S) from Yersinia pseud... -

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Basic information

Entry
Database: PDB / ID: 6yhk
TitleCrystal structure of full-length CNFy (C866S) from Yersinia pseudotuberculosis
ComponentsCytotoxic necrotizing factor
KeywordsTOXIN / CNF / cytotoxic necrotizing factor / deamidase / RhoA modification / RhoA activation / putative ADP-ribosyltransferase
Function / homologyCytotoxic necrotizing factor, Rho-activating domain / Domain of unknown function DUF4765 / Cytotoxic necrotizing factor, Rho-activating domain superfamily / Rho-activating domain of cytotoxic necrotizing factor / Domain of unknown function (DUF4765) / Domain of unknown function DUF6543 / Family of unknown function (DUF6543) / Cytotoxic necrotizing factor-like, catalytic / Cytotoxic necrotizing factor
Function and homology information
Biological speciesYersinia pseudotuberculosis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.7 Å
Model detailsStructure comprises residues 1-1014, active site cysteine 866 has been mutated to serine
AuthorsLukat, P. / Gazdag, E.M. / Heidler, T.V. / Blankenfeldt, W.
CitationJournal: Embo J. / Year: 2021
Title: Crystal structure of bacterial cytotoxic necrotizing factor CNF Y reveals molecular building blocks for intoxication.
Authors: Chaoprasid, P. / Lukat, P. / Muhlen, S. / Heidler, T. / Gazdag, E.M. / Dong, S. / Bi, W. / Ruter, C. / Kirchenwitz, M. / Steffen, A. / Jansch, L. / Stradal, T.E.B. / Dersch, P. / Blankenfeldt, W.
History
DepositionMar 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 30, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 24, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytotoxic necrotizing factor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,82011
Polymers114,9201
Non-polymers90010
Water1,00956
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1760 Å2
ΔGint-128 kcal/mol
Surface area40840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.153, 180.416, 220.761
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number24
Space group name H-MI212121

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Components

#1: Protein Cytotoxic necrotizing factor


Mass: 114920.227 Da / Num. of mol.: 1 / Fragment: full-length CNFy / Mutation: C866S
Source method: isolated from a genetically manipulated source
Details: C866S variant of full-length CNFy from Y. pseudotuberculosis
Source: (gene. exp.) Yersinia pseudotuberculosis (bacteria) / Gene: cnf / Plasmid: pCOLA-Duet-1
Details (production host): pCOLA-Duet-1 has been modified with sequence encoding for N-terminal Strep-tag II and TEV protease cleavage site
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0N9JNY6
#2: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 56 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 3.04 Å3/Da / Density % sol: 59.53 % / Mosaicity: 0.17 °
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 7.1
Details: 1.4 M ammonium sulfate, 0.13 M lithium acetate, 0.1 M HEPES/NaOH pH 7.1

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Apr 28, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.7→47.08 Å / Num. obs: 38996 / % possible obs: 99.9 % / Redundancy: 21.4 % / CC1/2: 1 / Rmerge(I) obs: 0.106 / Rpim(I) all: 0.023 / Rrim(I) all: 0.108 / Net I/σ(I): 23.3 / Num. measured all: 835740 / Scaling rejects: 1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
2.7-2.82221.80546980.7610.3921.84799.5
9.35-47.0820.60.028104110.0060.02899.3

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
Aimless0.5.32data scaling
PHASERphasing
PHENIX1.14rc1_3177refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Models of N-terminal and C-terminal domains

Resolution: 2.7→47.078 Å / SU ML: 0.38 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 26.84
RfactorNum. reflection% reflection
Rfree0.2419 1991 5.11 %
Rwork0.2048 --
obs0.2067 38988 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 208.55 Å2 / Biso mean: 85.66 Å2 / Biso min: 48.65 Å2
Refinement stepCycle: final / Resolution: 2.7→47.078 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7819 0 46 56 7921
Biso mean--109.74 67.6 -
Num. residues----994
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
2.7-2.76750.36511360.31132600
2.7675-2.84240.32621470.29732573
2.8424-2.9260.31341480.28682616
2.926-3.02040.33351420.27892602
3.0204-3.12830.28011650.27152604
3.1283-3.25360.31941300.25842605
3.2536-3.40160.33231320.24252658
3.4016-3.58090.25831520.22072609
3.5809-3.80510.24071530.21242599
3.8051-4.09880.26821460.1892658
4.0988-4.5110.19021290.15942653
4.511-5.1630.18791240.1612683
5.163-6.50210.21821340.19642719
6.5021-47.0780.21251530.19212818
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.39330.30170.27272.93990.49510.2151-0.16320.25670.0802-0.19120.1591-0.56050.17520.67840.00010.86180.00430.06961.26850.07190.81845.8479-15.373824.9445
23.0002-0.01450.09963.10070.34943.11340.05460.10750.0265-0.5367-0.1499-0.27110.05440.81910.00010.97480.10290.06621.26790.00360.66251.1665-22.95815.2794
30.9987-0.1035-0.55641.504-0.13413.193-0.0835-0.00440.0566-0.03490.0370.1971-0.03040.001800.533-0.0324-0.08940.5103-0.01380.7693-14.726628.118141.1323
41.5232-0.10720.27971.90790.26891.40060.0130.0977-0.1246-0.09620.02940.13870.2063-0.079100.7015-0.0742-0.05570.51830.00960.6964-13.135414.347631.9438
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 686 through 774 )A686 - 774
2X-RAY DIFFRACTION2chain 'A' and (resid 775 through 1014 )A775 - 1014
3X-RAY DIFFRACTION3chain 'A' and (resid -2 through 302 )A-2 - 302
4X-RAY DIFFRACTION4chain 'A' and (resid 303 through 685 )A303 - 685

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