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- PDB-8at0: PotF with mutation D247K -

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Basic information

Entry
Database: PDB / ID: 8at0
TitlePotF with mutation D247K
ComponentsPutrescine-binding periplasmic protein PotF
KeywordsTRANSPORT PROTEIN / Periplasmic-binding protein / Non-binding variant
Function / homology
Function and homology information


putrescine binding / putrescine transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / outer membrane-bounded periplasmic space / membrane
Similarity search - Function
Spermidine/putrescine-binding periplasmic protein / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
(2R)-1-methoxypropan-2-amine / MALONATE ION / (2~{S})-1-(2-methoxyethoxy)propan-2-amine / Putrescine-binding periplasmic protein PotF
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsKroeger, P. / Shanmugaratnam, S. / Hocker, B.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)HO4022/2-3 Germany
CitationJournal: To Be Published
Title: A fluorescent biosensor for the visualization of Agmatine
Authors: Kroeger, P. / Stiel, A.C. / Stueven, B. / Shanmugaratnam, S. / Schoch, S. / Wachten, D. / Hocker, B.
History
DepositionAug 22, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putrescine-binding periplasmic protein PotF
B: Putrescine-binding periplasmic protein PotF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,76216
Polymers78,3692
Non-polymers1,39414
Water5,513306
1
A: Putrescine-binding periplasmic protein PotF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,9999
Polymers39,1841
Non-polymers8158
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Putrescine-binding periplasmic protein PotF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,7637
Polymers39,1841
Non-polymers5796
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)70.700, 70.700, 272.640
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Putrescine-binding periplasmic protein PotF


Mass: 39184.434 Da / Num. of mol.: 2 / Mutation: D247K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: potF, b0854, JW0838 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P31133

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Non-polymers , 6 types, 320 molecules

#2: Chemical ChemComp-ONT / (2~{S})-1-(2-methoxyethoxy)propan-2-amine / Jeffamine


Mass: 133.189 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H15NO2
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-JFN / (2R)-1-methoxypropan-2-amine / jeffamine


Mass: 89.136 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C4H11NO
#5: Chemical ChemComp-MLI / MALONATE ION


Mass: 102.046 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H2O4
#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 306 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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: 2.51 Å3/Da / Density % sol: 50.99 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 9
Details: 2.4 M AmSO4, 0.1 M Bicine pH 9, 10% Jeffamine M600 pH7.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 7, 2021
RadiationMonochromator: DCM Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 2→45.55 Å / Num. obs: 54785 / % possible obs: 99.9 % / Redundancy: 11 % / Biso Wilson estimate: 39.35 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.181 / Rpim(I) all: 0.057 / Rrim(I) all: 0.19 / Net I/σ(I): 11.2
Reflection shellResolution: 2→2.07 Å / Redundancy: 11.5 % / Rmerge(I) obs: 2.98 / Mean I/σ(I) obs: 0.7 / Num. unique obs: 5385 / CC1/2: 0.256 / CC star: 0.639 / Rpim(I) all: 0.918 / Rrim(I) all: 3.12 / % possible all: 100

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Processing

Software
NameVersionClassification
XDS20210323data reduction
XSCALE20210323data scaling
PHENIX1.20.1_4487refinement
Coot0.9.6model building
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1A99

1a99


Resolution: 2→45.55 Å / SU ML: 0.2648 / Cross valid method: FREE R-VALUE / Phase error: 25.75
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2431 2101 3.83 %
Rwork0.2015 52684 -
obs0.203 54785 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 45.95 Å2
Refinement stepCycle: LAST / Resolution: 2→45.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5362 0 88 306 5756
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00315668
X-RAY DIFFRACTIONf_angle_d0.57447712
X-RAY DIFFRACTIONf_chiral_restr0.0444848
X-RAY DIFFRACTIONf_plane_restr0.0042994
X-RAY DIFFRACTIONf_dihedral_angle_d12.1542081
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.050.31991370.30893418X-RAY DIFFRACTION99.03
2.05-2.10.38821380.30473459X-RAY DIFFRACTION99.97
2.1-2.150.33431370.30383453X-RAY DIFFRACTION99.89
2.15-2.220.351400.26993508X-RAY DIFFRACTION100
2.22-2.290.3081370.26123427X-RAY DIFFRACTION100
2.29-2.370.28761380.25263475X-RAY DIFFRACTION99.94
2.37-2.460.30061380.23753453X-RAY DIFFRACTION99.97
2.46-2.580.3061380.23793468X-RAY DIFFRACTION100
2.58-2.710.31121400.24083525X-RAY DIFFRACTION99.97
2.71-2.880.31721390.23963476X-RAY DIFFRACTION99.97
2.88-3.110.27031400.22173518X-RAY DIFFRACTION99.97
3.11-3.420.22831410.20643538X-RAY DIFFRACTION99.97
3.42-3.910.21631420.17283561X-RAY DIFFRACTION99.97
3.91-4.930.1841440.1433601X-RAY DIFFRACTION100
4.93-45.550.18581520.17323804X-RAY DIFFRACTION99.82
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)
12.695482746720.3133226728060.6568635857221.63785721310.3873469853470.6223934573780.0110148536091-0.0606605407996-0.002888343174280.0867685785915-0.05402248983830.197549099626-0.156637792243-0.1552424377830.03838375342630.3985128433080.0797959503637-0.02314923717040.2757546619410.0503166267850.289977716267-6.3523207614721.3029552529-71.2107001922
21.743218397580.399606262564-1.086358803211.21017510954-0.2320222582041.88858879070.01758787191-0.06463575851920.1426734853280.0655743959205-0.0318050076991-0.04432207377780.0455692417036-0.07525382743410.008367434778660.3937597930770.0513088164862-0.003347613495130.194933826211-0.04239535520530.302952301216-13.8120416599-9.93087175816-65.4369925623
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth seq-ID: 29 - 369 / Label seq-ID: 1 - 341

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-ID
11(chain 'A' and resid 29 through 369)AA
22(chain 'B' and resid 29 through 369)BC

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