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- PDB-5a6s: Crystal structure of the CTP1L endolysin reveals how its activity... -

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Basic information

Entry
Database: PDB / ID: 5a6s
TitleCrystal structure of the CTP1L endolysin reveals how its activity is regulated by a secondary translation product
Components(ENDOLYSINLysin) x 2
KeywordsSTRUCTURAL PROTEIN / ENDOLYSIN / SECONDARY TRANSLATION PRODUCT / BACTERIOPHAGE
Function / homology
Function and homology information


peptidoglycan catabolic process / cell wall macromolecule catabolic process / lysozyme / lysozyme activity
Similarity search - Function
Rossmann fold - #12090 / Glycosyl hydrolase family 25 (GH25) domain profile. / Glycoside hydrolase, family 25 / Glycosyl hydrolases family 25 / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesCLOSTRIDIUM PHAGE PHICTP1 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 Å
AuthorsDunne, M. / Leicht, S. / Krichel, B. / Mertens, H.D.T. / Thompson, A. / Krijgsveld, J. / Svergun, D.I. / GomezTorres, N. / Garde, S. / Uetrecht, C. ...Dunne, M. / Leicht, S. / Krichel, B. / Mertens, H.D.T. / Thompson, A. / Krijgsveld, J. / Svergun, D.I. / GomezTorres, N. / Garde, S. / Uetrecht, C. / Narbad, A. / Mayer, M.J. / Meijers, R.
CitationJournal: J Biol Chem / Year: 2016
Title: Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product.
Authors: Matthew Dunne / Stefan Leicht / Boris Krichel / Haydyn D T Mertens / Andrew Thompson / Jeroen Krijgsveld / Dmitri I Svergun / Natalia Gómez-Torres / Sonia Garde / Charlotte Uetrecht / Arjan ...Authors: Matthew Dunne / Stefan Leicht / Boris Krichel / Haydyn D T Mertens / Andrew Thompson / Jeroen Krijgsveld / Dmitri I Svergun / Natalia Gómez-Torres / Sonia Garde / Charlotte Uetrecht / Arjan Narbad / Melinda J Mayer / Rob Meijers /
Abstract: Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular ...Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.
History
DepositionJul 1, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 30, 2015Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2016Group: Database references
Revision 1.2Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_sheet / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_sheet.number_strands / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ENDOLYSIN
B: ENDOLYSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,5197
Polymers41,9082
Non-polymers6115
Water8,647480
1
A: ENDOLYSIN
B: ENDOLYSIN
hetero molecules

A: ENDOLYSIN
B: ENDOLYSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,03814
Polymers83,8174
Non-polymers1,22110
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_556y,x,-z+11
Buried area8420 Å2
ΔGint-50.1 kcal/mol
Surface area31650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)136.197, 136.197, 56.457
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein ENDOLYSIN / Lysin


Mass: 32877.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM PHAGE PHICTP1 (virus) / Strain: CTP1L / Description: TARGET AGAINST CLOSTRIDIUM TYROBUTYRICUM / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: D9ZNF3
#2: Protein ENDOLYSIN / Lysin


Mass: 9031.121 Da / Num. of mol.: 1
Fragment: TRUNCATED CELL WALL BINDING DOMAIN, RESIDUES 195-274
Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) CLOSTRIDIUM PHAGE PHICTP1 (virus) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: D9ZNF3

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Non-polymers , 4 types, 485 molecules

#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400 / Polyethylene glycol


Mass: 238.278 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 480 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHIS CHAIN IS A TRUNCATED CELL WALL BINDING DOMAIN THAT IS THE RESULT OF SECONDARY TRANSLATION ...THIS CHAIN IS A TRUNCATED CELL WALL BINDING DOMAIN THAT IS THE RESULT OF SECONDARY TRANSLATION WITHIN THE ENDOLYSIN GENE. THE VAL195 IS REPLACED BY A METHIONINE

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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: 3.12 Å3/Da / Density % sol: 60.62 % / Description: NONE
Crystal growDetails: 5-10 % PEG 8000, 20 MM TRIS PH 8.0

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.98
DetectorType: ADSC CCD / Detector: CCD / Details: KB MIRRORS
RadiationMonochromator: SI 1 1 1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.9→30 Å / Num. obs: 40146 / % possible obs: 100 % / Observed criterion σ(I): 0 / Redundancy: 8.2 % / Rmerge(I) obs: 0.15 / Net I/σ(I): 9.6
Reflection shellResolution: 1.9→2 Å / Redundancy: 8 % / Rmerge(I) obs: 0.75 / Mean I/σ(I) obs: 2.6 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.7.0029refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRIES 1JFX AND 2NW0 HYBRID MODEL

1jfx

2nw0


Resolution: 1.9→19.9 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.943 / SU B: 2.55 / SU ML: 0.075 / Cross valid method: THROUGHOUT / ESU R: 0.11 / ESU R Free: 0.114 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.20468 2132 5 %RANDOM
Rwork0.16196 ---
obs0.16412 40146 99.87 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 26.486 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å20 Å20 Å2
2---0.05 Å20 Å2
3---0.09 Å2
Refinement stepCycle: LAST / Resolution: 1.9→19.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2780 0 39 480 3299
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.022869
X-RAY DIFFRACTIONr_bond_other_d0.0010.022692
X-RAY DIFFRACTIONr_angle_refined_deg1.81.9513881
X-RAY DIFFRACTIONr_angle_other_deg0.86536173
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1515350
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.17525.149134
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.80815471
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.649158
X-RAY DIFFRACTIONr_chiral_restr0.1270.2435
X-RAY DIFFRACTIONr_gen_planes_refined0.010.023275
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02681
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_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.901→1.95 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.324 151 -
Rwork0.273 2913 -
obs--100 %

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