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- PDB-9g1d: Fragment screening of FosAKP, room-temperature structure in compl... -

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Basic information

Entry
Database: PDB / ID: 9g1d
TitleFragment screening of FosAKP, room-temperature structure in complex with fragment F2X-entry E04
ComponentsFosfomycin resistance protein
KeywordsTRANSFERASE / antibiotic resistance / fosfomycin / fragment screening
Function / homology
Function and homology information


: / Glyoxalase/fosfomycin resistance/dioxygenase domain / Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily / Vicinal oxygen chelate (VOC) domain / Vicinal oxygen chelate (VOC) domain profile. / Glyoxalase/Bleomycin resistance protein/Dihydroxybiphenyl dioxygenase
Similarity search - Domain/homology
: / : / Fosfomycin resistance protein
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsGuenther, S. / Galchenkova, M. / Fischer, P. / Reinke, P.Y.A. / Falke, S. / Thekku Veedu, S. / Rodrigues, A.C. / Senst, J. / Meents, A.
Funding support Germany, 5items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research16GW0277 Germany
German Federal Ministry for Education and Research13K22CHB Germany
Helmholtz AssociationFISCOV Germany
Helmholtz AssociationSFragX Germany
Helmholtz AssociationHIR3X Germany
CitationJournal: To Be Published
Title: Room temperature X-ray fragment screening with serial crystallography
Authors: Guenther, S. / Meents, A.
History
DepositionJul 10, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fosfomycin resistance protein
B: Fosfomycin resistance protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,8895
Polymers32,6192
Non-polymers2703
Water4,324240
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5740 Å2
ΔGint-41 kcal/mol
Surface area12970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.730, 91.650, 45.340
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Space group name HallP22ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x+1/2,y+1/2,-z
#4: -x,-y,z

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Components

#1: Protein Fosfomycin resistance protein


Mass: 16309.347 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: KPNJ1_04856 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0E1CQ35
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-VNV / 3-phenyl-1,2-oxazol-5-amine


Mass: 160.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H8N2O / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 240 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.45 Å3/Da / Density % sol: 49.74 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 25 mg/mL FosAKP in 10 mM Hepes, pH 7.5, 75 mM NaCl was supplemented with 6 mM MnCl2 and mixed with an equal volume of 16% (w/v) PEG3350, 0.25 M MgCl2, 0.2 M KBr, 0.1 M BisTris, pH 5.5 and ...Details: 25 mg/mL FosAKP in 10 mM Hepes, pH 7.5, 75 mM NaCl was supplemented with 6 mM MnCl2 and mixed with an equal volume of 16% (w/v) PEG3350, 0.25 M MgCl2, 0.2 M KBr, 0.1 M BisTris, pH 5.5 and 1/10 volume of seed stock in 26% (w/v) PEG3350, 0.25 M MgCl2, 0.2 M KBr, 0.1 M BisTris, pH 5.5. Approximately of this solution 14 uL were added per window of the fixed-target chip. The sample holder was then inserted for into a 3D-printed crystal growth chamber with 3 mL of precipitant solution in the bottom for vapor-diffusion crystallization and incubated at 20C. For fragment application sample holders were removed from the crystal growth chamber and excess precipitant was removed by blotting through the micropores of the membranes, before 10 uL of fragment solution at a concentration of 25 mM in 5% DMSO were pipetted to the crystals in the individual compartments. Sample holders were then placed back into the growths vessel and incubated for 24h. Before data collection sample holder was removed from the crystal growth chamber and excess precipitant was removed by blotting through the micropores of the membranes, before 10 uL of crystallization solution with 5% DMSO were pipetted to the crystals in the individual compartments. Sample holders were then placed back into the growths vessel and incubated for 24h. Before data collection blotting was repeated for removal of excess liquid in order to minimize background scattering. Sample holders were then equipped with a protective cover to prevent them from drying-out and stored in a humid atmosphere. Compound addition and liquid removal were conducted in a glove box with >95% rel. humidity.
PH range: 5.5

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Data collection

DiffractionMean temperature: 296.15 K
Ambient temp details: Temperature and relative humidity was controlled during experiment using a custom-build setup.
Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P09 HiPhaX / Wavelength: 0.7749 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Dec 20, 2023 / Details: CRL
RadiationMonochromator: Si111 DCM / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.7749 Å / Relative weight: 1
ReflectionResolution: 1.4→91.5 Å / Num. obs: 59715 / % possible obs: 100 % / Redundancy: 191.5 % / Biso Wilson estimate: 18.64 Å2 / CC1/2: 0.9507 / CC star: 0.9873 / R split: 0.1724 / Net I/σ(I): 4.8
Reflection shellResolution: 1.4→1.45 Å / Redundancy: 81.1 % / Mean I/σ(I) obs: 1.07 / Num. unique obs: 5872 / CC1/2: 0.585 / CC star: 0.859 / R split: 0.719 / % possible all: 100
Serial crystallography sample deliveryDescription: fixed target using Kapton chip / Method: fixed target
Serial crystallography sample delivery fixed targetDescription: Crystals were directly grown on the chip surface. Excess liquid was removed by blotting in an glove box to maintain rel. humidity levels >95%.
Motion control: Roadrunner Goniometer
Sample dehydration prevention: Temperature and rel. humdity levels around sample were tightly controlled with custom setup. Furthermore crystals were protected from dehyration using a mylar cover for the chip.
Sample holding: micro-perforated kapton / Sample unit size: 25 µm / Support base: kinematic mount
Serial crystallography data reductionFrame hits: 35699 / Frames indexed: 20239 / Frames total: 35904 / Lattices indexed: 28878

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Processing

Software
NameVersionClassification
PHENIX1.20-4459_9999phasing
PHENIX1.20-4459_9999refinement
CrystFEL0.10.1data reduction
CrystFEL0.10.1data scaling
Coot0.9.8.92model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→56.49 Å / SU ML: 0.2368 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 19.838
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1997 2901 4.89 %
Rwork0.1736 56466 -
obs0.1748 59367 99.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 22.81 Å2
Refinement stepCycle: LAST / Resolution: 1.4→56.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2144 0 14 240 2398
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01072402
X-RAY DIFFRACTIONf_angle_d1.0643295
X-RAY DIFFRACTIONf_chiral_restr0.0897343
X-RAY DIFFRACTIONf_plane_restr0.011445
X-RAY DIFFRACTIONf_dihedral_angle_d5.2042353
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4-1.420.50951580.46272565X-RAY DIFFRACTION97.35
1.42-1.450.39541470.38912575X-RAY DIFFRACTION97.91
1.45-1.470.39551450.33772625X-RAY DIFFRACTION98.37
1.47-1.50.3481460.27892628X-RAY DIFFRACTION98.86
1.5-1.530.24891320.22752634X-RAY DIFFRACTION99.21
1.53-1.570.25051330.19592687X-RAY DIFFRACTION99.61
1.57-1.60.24151290.17922675X-RAY DIFFRACTION99.82
1.6-1.640.21871450.17142645X-RAY DIFFRACTION99.68
1.64-1.690.19471180.17982685X-RAY DIFFRACTION99.64
1.69-1.740.21121280.17922701X-RAY DIFFRACTION99.89
1.74-1.790.21621170.1932698X-RAY DIFFRACTION99.96
1.79-1.860.23121200.17882694X-RAY DIFFRACTION99.89
1.86-1.930.19751130.15832707X-RAY DIFFRACTION99.75
1.93-2.020.2031550.13212672X-RAY DIFFRACTION99.89
2.02-2.130.15191500.13072701X-RAY DIFFRACTION100
2.13-2.260.16861440.13862692X-RAY DIFFRACTION100
2.26-2.430.1671430.15062714X-RAY DIFFRACTION100
2.43-2.680.1721350.15772736X-RAY DIFFRACTION100
2.68-3.070.20991470.17492740X-RAY DIFFRACTION100
3.07-3.860.18421550.1592769X-RAY DIFFRACTION100
3.86-56.490.1811410.18122923X-RAY DIFFRACTION99.87

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