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- PDB-5u89: Crystal structure of a cross-module fragment from the dimodular N... -

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Basic information

Entry
Database: PDB / ID: 5u89
TitleCrystal structure of a cross-module fragment from the dimodular NRPS DhbF
Components
  • Amino acid adenylation domain protein
  • MbtH domain protein
KeywordsHYDROLASE/INHIBITOR / nonribosomal peptide synthetase / MbtH-like protein / mechanism-based inhibitor / megaenzyme / HYDROLASE-INHIBITOR complex
Function / homology
Function and homology information


biosynthetic process / hydrolase activity, acting on ester bonds / phosphopantetheine binding
Similarity search - Function
Rubredoxin-like / Structural Genomics, Unknown Function 30-nov-00 1gh9 Mol_id / MbtH-like protein / MbtH-like domain / MbtH-like domain superfamily / MbtH-like protein / MbtH-like protein / Polyketide synthase, thioesterase domain / Thioesterase / Thioesterase ...Rubredoxin-like / Structural Genomics, Unknown Function 30-nov-00 1gh9 Mol_id / MbtH-like protein / MbtH-like domain / MbtH-like domain superfamily / MbtH-like protein / MbtH-like protein / Polyketide synthase, thioesterase domain / Thioesterase / Thioesterase / Thioesterase domain / Condensation domain / Condensation domain / Amino acid adenylation domain / ANL, N-terminal domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / AMP-binding, conserved site / Chloramphenicol acetyltransferase-like domain superfamily / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase / AMP-binding enzyme / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Chem-MJ8 / Amino acid adenylation domain protein / MbtH domain protein / MbtH domain protein / Amino acid adenylation domain protein
Similarity search - Component
Biological speciesGeobacillus sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 3.075 Å
AuthorsTarry, M.J. / Schmeing, T.M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-148472 Canada
CitationJournal: Structure / Year: 2017
Title: X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture.
Authors: Michael J Tarry / Asfarul S Haque / Khanh Huy Bui / T Martin Schmeing /
Abstract: Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little ...Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture.
History
DepositionDec 14, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2017Provider: repository / Type: Initial release
Revision 1.1May 17, 2017Group: Database references
Revision 1.2Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Amino acid adenylation domain protein
B: MbtH domain protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,3353
Polymers130,5912
Non-polymers7441
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2250 Å2
ΔGint-17 kcal/mol
Surface area46650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)119.510, 119.510, 589.647
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein Amino acid adenylation domain protein / Bacillibactin synthetase subunit F


Mass: 121345.070 Da / Num. of mol.: 1 / Fragment: UNP residues 437-1504
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus sp. (bacteria) / Strain: Y4.1MC1 / Gene: GY4MC1_0171 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0F6BHX2, UniProt: A0A7U3YCA6*PLUS
#2: Protein MbtH domain protein


Mass: 9246.290 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus sp. (bacteria) / Strain: Y4.1MC1 / Gene: GY4MC1_0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0F6BHX3, UniProt: A0A7U3YBY3*PLUS
#3: Chemical ChemComp-MJ8 / 5'-({[(2R)-3-amino-2-{[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl]sulfanyl}propyl]sulfonyl}amino)-5'-deoxyadenosine


Mass: 743.747 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H42N9O12PS2

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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: 4.65 Å3/Da / Density % sol: 73.57 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 1.6 M ammonium sulfate, 0.1 M Bis-Tris pH 5, 2 mM EDTA, 0.008% (v/v) octyl-beta-D galactoside

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CLSI / Beamline: 08ID-1 / Wavelength: 0.9795 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Oct 7, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.075→58.56 Å / Num. obs: 47171 / % possible obs: 98.56 % / Redundancy: 2 % / Rmerge(I) obs: 0.06869 / Rsym value: 0.06869 / Net I/σ(I): 5.04

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
iMOSFLMdata reduction
SCALAdata scaling
PHENIXphasing
RefinementResolution: 3.075→58.564 Å / SU ML: 0.44 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2588 2295 4.87 %
Rwork0.2163 --
obs0.2184 47160 98.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.075→58.564 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8060 0 47 0 8107
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0038308
X-RAY DIFFRACTIONf_angle_d0.61611415
X-RAY DIFFRACTIONf_dihedral_angle_d11.4872847
X-RAY DIFFRACTIONf_chiral_restr0.0241362
X-RAY DIFFRACTIONf_plane_restr0.0031472
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.0753-3.14210.40051240.37222271X-RAY DIFFRACTION82
3.1421-3.21520.39221320.32812754X-RAY DIFFRACTION100
3.2152-3.29560.36131430.31382773X-RAY DIFFRACTION100
3.2956-3.38470.31951690.29452751X-RAY DIFFRACTION100
3.3847-3.48430.3671490.28092775X-RAY DIFFRACTION100
3.4843-3.59670.27061150.25632824X-RAY DIFFRACTION100
3.5967-3.72530.29271330.24392811X-RAY DIFFRACTION100
3.7253-3.87440.24651490.22592790X-RAY DIFFRACTION100
3.8744-4.05070.24431600.20412784X-RAY DIFFRACTION100
4.0507-4.26420.22791570.18722824X-RAY DIFFRACTION100
4.2642-4.53130.19241270.1722823X-RAY DIFFRACTION100
4.5313-4.8810.21891430.16552832X-RAY DIFFRACTION100
4.881-5.37190.19931350.17722877X-RAY DIFFRACTION100
5.3719-6.14840.27761530.20692880X-RAY DIFFRACTION99
6.1484-7.74360.24441510.21942945X-RAY DIFFRACTION99
7.7436-58.57480.25011550.19573151X-RAY DIFFRACTION98
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)
13.51910.4268-0.37151.1516-0.5791.59610.1180.1826-0.1432-0.1408-0.1023-0.03270.04420.0613-0.05660.36370.01460.00210.62940.24790.5846-44.991243.2241-1.415
28.8272-0.93730.66913.82540.64812.35490.29880.3960.06640.2220.0507-0.01090.01540.1072-0.21450.4601-0.05750.1760.99630.58141.1434-63.913669.1544-10.4465
35.19573.56171.21473.76520.84712.838-0.14860.91580.7153-0.10750.596-0.011-0.4264-0.0485-0.34130.4259-0.03490.15721.19340.59091.0805-66.179871.2076-15.0332
45.3971-0.17711.70752.7031-0.48994.0956-0.0693-0.40330.2666-0.10760.30730.48740.01-0.1418-0.19890.56340.08270.01090.34020.13040.3775-40.1025104.8971-35.5572
51.8843-0.89820.30582.2024-0.57831.06610.021-0.23490.0074-0.12140.10610.0403-0.0381-0.077-0.12060.38060.018-0.01340.49820.14510.4057-29.415490.658-32.4123
65.24090.3355-0.51422.68511.88763.86690.4771-0.49320.25680.2641-0.3299-0.1417-0.18980.3935-0.070.4317-0.08950.13390.66370.26390.754-62.831845.867920.7599
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 438:853)
2X-RAY DIFFRACTION2(chain A and resid 854:902)
3X-RAY DIFFRACTION3(chain A and resid 912:957)
4X-RAY DIFFRACTION4(chain A and resid 1058:1114)
5X-RAY DIFFRACTION5(chain A and resid 1118:1494)
6X-RAY DIFFRACTION6(chain B and resid 6:69)

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