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- PDB-6vhy: NpsA-ThdA, an artificially fused Adenylation-PCP di-domain NRPS f... -

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Basic information

Entry
Database: PDB / ID: 6vhy
TitleNpsA-ThdA, an artificially fused Adenylation-PCP di-domain NRPS from Klebsiella oxytoca
ComponentsNpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
KeywordsBIOSYNTHETIC PROTEIN / adenylation / tilivalline / tilimycin / NRPS / nonribosomal peptide synthetase
Function / homology
Function and homology information


ANL, N-terminal domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / AMP-dependent synthetase/ligase / AMP-binding enzyme / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Chem-QXD / Non-ribosomal peptide synthetase / Thioester reductase
Similarity search - Component
Biological speciesKlebsiella oxytoca (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsKreitler, D.F. / Gulick, A.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM116957 United States
CitationJournal: Acs Infect Dis. / Year: 2020
Title: Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic PathogenKlebsiella oxytoca.
Authors: Alexander, E.M. / Kreitler, D.F. / Guidolin, V. / Hurben, A.K. / Drake, E. / Villalta, P.W. / Balbo, S. / Gulick, A.M. / Aldrich, C.C.
History
DepositionJan 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.name
Revision 1.2Oct 11, 2023Group: 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 / software
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
B: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
C: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
D: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,4778
Polymers256,2504
Non-polymers3,2274
Water181
1
A: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
C: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,7394
Polymers128,1252
Non-polymers1,6142
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5830 Å2
ΔGint-31 kcal/mol
Surface area44710 Å2
MethodPISA
2
B: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
D: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,7394
Polymers128,1252
Non-polymers1,6142
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5190 Å2
ΔGint-26 kcal/mol
Surface area41780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.540, 98.721, 118.754
Angle α, β, γ (deg.)95.331, 96.337, 106.508
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein
NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein


Mass: 64062.523 Da / Num. of mol.: 4 / Mutation: E312A,E313A,Q314A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Gene: AGF18_11095, AGF18_11090 / Plasmid: pET15 / Details (production host): N-term TEV tag / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U4DY99, UniProt: A0A2U4DNX7
#2: Chemical
ChemComp-QXD / 5'-deoxy-5'-({[(2R)-2-{[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl]sulfanyl}-2-(3-hydroxyphenyl)ethyl]sulfonyl}amino)adenosine


Mass: 806.801 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: C29H43N8O13PS2 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.53 Å3/Da / Density % sol: 51.39 % / Description: plates
Crystal growTemperature: 287 K / Method: microbatch
Details: protein solution (19 mg/mL): 10 mM HEPES pH 8.0, 25 mM NaCl, 0.4 mM TCEP, 2.5 mM MgCl2, 5% v/v glycerol, 1.2 mM 3-hydroxybenzoyl-AVS, well solution: (0.2 M sodium formate, 20% w/v PEG3350, ...Details: protein solution (19 mg/mL): 10 mM HEPES pH 8.0, 25 mM NaCl, 0.4 mM TCEP, 2.5 mM MgCl2, 5% v/v glycerol, 1.2 mM 3-hydroxybenzoyl-AVS, well solution: (0.2 M sodium formate, 20% w/v PEG3350, microbatch drop: 1.2 uL protein solution, 1 uL well solution (under paraffin oil)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 3→93.84 Å / Num. obs: 45795 / % possible obs: 90.9 % / Redundancy: 7.6 % / Biso Wilson estimate: 74.91 Å2 / CC1/2: 0.997 / Rpim(I) all: 0.045 / Rrim(I) all: 0.124 / Rsym value: 0.116 / Net I/σ(I): 12.2
Reflection shellResolution: 3→3.05 Å / Redundancy: 7.7 % / Mean I/σ(I) obs: 2.3 / Num. unique obs: 2224 / CC1/2: 0.742 / Rpim(I) all: 0.338 / Rrim(I) all: 0.945 / Rsym value: 0.882 / % possible all: 87.7

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Processing

Software
NameVersionClassification
PHENIX1.15_3459refinement
XDSdata reduction
Aimless0.7.4data scaling
autoPROC1.0.5 (20190301)data scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6vhu

6vhu


Resolution: 3→47.61 Å / SU ML: 0.3855 / Cross valid method: FREE R-VALUE / σ(F): 2 / Phase error: 26.989
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2604 2238 4.89 %
Rwork0.2141 43529 -
obs0.2163 45767 90.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 83.62 Å2
Refinement stepCycle: LAST / Resolution: 3→47.61 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15234 0 201 1 15436
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00615750
X-RAY DIFFRACTIONf_angle_d0.73121535
X-RAY DIFFRACTIONf_chiral_restr0.05192577
X-RAY DIFFRACTIONf_plane_restr0.0052760
X-RAY DIFFRACTIONf_dihedral_angle_d14.31239329
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.060.34441360.28032625X-RAY DIFFRACTION86.8
3.06-3.130.30511130.26032571X-RAY DIFFRACTION85.78
3.13-3.210.28831570.25972835X-RAY DIFFRACTION94.21
3.21-3.30.29941430.2542794X-RAY DIFFRACTION93.77
3.3-3.40.28191460.24262800X-RAY DIFFRACTION93.41
3.4-3.510.26871410.232770X-RAY DIFFRACTION92.59
3.51-3.630.29861450.21862694X-RAY DIFFRACTION90.47
3.63-3.780.25251590.22082587X-RAY DIFFRACTION87.7
3.78-3.950.23611350.21212711X-RAY DIFFRACTION89.36
3.95-4.160.22861540.1932787X-RAY DIFFRACTION93.9
4.16-4.420.23611190.18992785X-RAY DIFFRACTION93.17
4.42-4.760.22481440.18472782X-RAY DIFFRACTION91.7
4.76-5.240.24141090.19852590X-RAY DIFFRACTION86.09
5.24-5.990.30371560.22632786X-RAY DIFFRACTION94.02
5.99-7.550.27491440.22592641X-RAY DIFFRACTION88.08
7.55-47.610.24551370.20182771X-RAY DIFFRACTION92.52
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)
11.334530420510.8551342575520.1845746656683.736226795450.4394148612561.585420893460.1070959154960.0348738573508-0.1423525772240.3649953245350.00277403432297-0.116341361919-0.173070706612-0.0184989143999-0.1005573384660.2957858230010.1022095407530.03937044854970.4321169358820.05064599128750.441587536058-29.147836015793.8565738994-31.6559141606
27.299011265521.941003768761.600098588878.348385586741.253060358080.7184071015410.340676943349-0.2979435755210.7453147397311.15526213411-0.1534435942090.889507733171-0.244472217964-0.404144977783-0.2457080497160.9576838560050.1620824534020.2391287570740.598534201601-0.05612450456710.467932309392-38.6323558361109.092702545-23.4712220627
33.07436723902-0.05608543756440.3776053943132.74246654504-0.0240913668054.550782192740.0739998506437-0.4583009238590.5233166712340.411322337721-0.1665512074180.00713027522114-0.4281630061770.4816883830070.08596688909170.609701789617-0.116073770374-0.0111609675090.635591706141-0.04734617868270.46153912569814.700296329347.8093946895-7.37871109139
42.235543768691.0955646154-0.2447394072244.54891026437-0.5042270772252.46112287485-0.2189043246230.05918479003460.00883600404083-0.4812158420240.2492747356720.177497936764-0.303224515228-0.087443279012-0.04668129206770.4589118838430.0615547982439-0.01720728059960.4260590363370.02137754136340.441612063525-3.4410966726886.91514120148.6208875074
51.047075743250.2512294974250.1405430526014.233226703611.456600548585.24652448560.0494470426982-0.128270486929-0.2557298525980.2114121379610.143509183890.3700994533410.43878307683-0.20751442276-0.1591167698360.2875358041060.02351911535680.03696320796360.409795590150.08498593597640.75265910560234.836416299227.062487992175.6758901223
63.425724833391.279622109360.2707002705878.174950264420.9141794725831.85483168857-0.04305914356490.0691386066867-0.2166112415640.4882516156110.05420316046180.2371638795510.3390934312950.0616137318259-0.004092496957730.3595735224790.16768919626-0.01450843400720.4144521247180.02932921445490.49067424497840.378540987829.110324175776.8959418943
71.095685520221.211989753180.1903771309992.70310173157-0.5754058098951.67716985751-0.110739580991-0.0129821814688-0.0181450823052-0.2336446860280.08815975693410.33624808864-0.0932716780831-0.1083239173140.01771202209430.2494562126850.1018048732240.001847162527920.4860789901420.008146414121360.58532600697637.386192595148.672672755968.1511786737
84.559592833970.892313341259-0.5196086664493.34647252901-0.5340536653041.22524821538-0.1267487082430.0838153995712-0.217234078578-0.1176282731790.260237435734-0.7895921152920.2511267799880.435696565065-0.1179948037610.3408081257570.1121614496090.05908602585550.551632638065-0.1054580843160.65877365495559.758640870343.808477977867.1282569322
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 5 through 376 )
2X-RAY DIFFRACTION2chain 'A' and (resid 377 through 416 )
3X-RAY DIFFRACTION3chain 'B' and (resid 4 through 425 )
4X-RAY DIFFRACTION4chain 'C' and (resid 4 through 392 )
5X-RAY DIFFRACTION5chain 'D' and (resid 6 through 125 )
6X-RAY DIFFRACTION6chain 'D' and (resid 126 through 192 )
7X-RAY DIFFRACTION7chain 'D' and (resid 193 through 314 )
8X-RAY DIFFRACTION8chain 'D' and (resid 315 through 412 )

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