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- PDB-7uwq: Klebsiella pneumoniae adenosine monophosphate nucleosidase -

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Basic information

Entry
Database: PDB / ID: 7uwq
TitleKlebsiella pneumoniae adenosine monophosphate nucleosidase
ComponentsAMP nucleosidase
KeywordsHYDROLASE / nucleosidase / AMP / salvage
Function / homology
Function and homology information


AMP nucleosidase / AMP nucleosidase activity / nucleoside metabolic process / AMP salvage / cytoplasm
Similarity search - Function
AMP nucleoside phosphorylase, N-terminal / AMP nucleoside phosphorylase, N-terminal domain superfamily / Bacterial AMP nucleoside phosphorylase N-terminus / AMP nucleosidase / Nucleoside phosphorylase domain / Phosphorylase superfamily / Nucleoside phosphorylase superfamily
Similarity search - Domain/homology
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsRichardson, B.C. / French, J.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM124898 United States
CitationJournal: PLoS One / Year: 2022
Title: Structure of Klebsiella pneumoniae adenosine monophosphate nucleosidase.
Authors: Brian C Richardson / Roger Shek / Wesley C Van Voorhis / Jarrod B French /
Abstract: Klebsiella pneumoniae is a bacterial pathogen that is increasingly responsible for hospital-acquired pneumonia and sepsis. Progressive development of antibiotic resistance has led to higher mortality ...Klebsiella pneumoniae is a bacterial pathogen that is increasingly responsible for hospital-acquired pneumonia and sepsis. Progressive development of antibiotic resistance has led to higher mortality rates and creates a need for novel treatments. Because of the essential role that nucleotides play in many bacterial processes, enzymes involved in purine and pyrimidine metabolism and transport are ideal targets for the development of novel antibiotics. Herein we describe the structure of K. pneumoniae adenosine monophosphate nucleosidase (KpAmn), a purine salvage enzyme unique to bacteria, as determined by cryoelectron microscopy. The data detail a well conserved fold with a hexameric overall structure and clear density for the putative active site residues. Comparison to the crystal structures of homologous prokaryotic proteins confirms the presence of many of the conserved structural features of this protein yet reveals differences in distal loops in the absence of crystal contacts. This first cryo-EM structure of an Amn enzyme provides a basis for future structure-guided drug development and extends the accuracy of structural characterization of this family of proteins beyond this clinically relevant organism.
History
DepositionMay 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AMP nucleosidase
B: AMP nucleosidase
C: AMP nucleosidase
D: AMP nucleosidase
E: AMP nucleosidase
F: AMP nucleosidase


Theoretical massNumber of molelcules
Total (without water)331,9586
Polymers331,9586
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "B"
d_3ens_1chain "A"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEUARGC1 - 433
d_21ens_1LEUARGB1 - 433
d_31ens_1LEUARGA1 - 433
d_41ens_1LEUARGD1 - 433
d_51ens_1LEUARGE1 - 433
d_61ens_1LEUARGF1 - 433

NCS oper:
IDCodeMatrixVector
1given(0.500411239514, 0.865787843745, -3.1491801E-5), (0.865787841782, -0.500411234933, 9.4740596E-5), (6.2652154E-5, -7.467653E-5, -0.999999995016)-25.3149094865, 43.8349847159, 115.539991931
2given(-0.503690106442, 0.863881881182, -0.00209094188407), (-0.863883586079, -0.50368480354, 0.00260161230198), (0.00119431007745, 0.0031167367504, 0.999994429772)46.3236711421, 166.813204512, -0.116585796718
3given(-0.99999309095, -0.000657065612889, 0.00365873171766), (-0.000667400499106, 0.999995789187, -0.00282421415163), (-0.00365686061742, -0.00282663647836, -0.999989318691)142.120683655, 0.264419593249, 115.80086727
4given(-0.500721258552, -0.865608387853, 0.000583192301137), (0.865606971756, -0.50072151258, -0.00159288629557), (0.00167083266949, -0.000292776708909, 0.999998561299)167.564179463, 43.8352086808, -0.0709989155511
5given(0.499618788321, -0.866244919906, -0.000897270341845), (-0.86624241448, -0.499619519701, 0.00210116373088), (-0.002268416185, -0.000272527249923, -0.999997390005)96.3884794334, 166.910483855, 115.663370304

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Components

#1: Protein
AMP nucleosidase


Mass: 55326.336 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Gene: amn, A7B01_08185, B4U61_13745, B5L96_10595, BL124_00007990, BN49_3643, BS595_23510, C3F39_24645, DDJ63_13085, DRB11_00280, EAO17_04140, FXN67_19000, G5637_19470, G7Z27_09550, GJJ12_013280, ...Gene: amn, A7B01_08185, B4U61_13745, B5L96_10595, BL124_00007990, BN49_3643, BS595_23510, C3F39_24645, DDJ63_13085, DRB11_00280, EAO17_04140, FXN67_19000, G5637_19470, G7Z27_09550, GJJ12_013280, GNE24_03025, GNG14_09410, GPZ86_08955, HV479_00330, NCTC11679_01882, NCTC13443_04974, NCTC13465_03257, NCTC204_04420, NCTC9128_08085, NCTC9617_06427, NCTC9637_02601, SAMEA3499874_04631, SAMEA3499901_02408, SAMEA3500057_04230, SAMEA3512100_00855, SAMEA3538828_00203, SAMEA3649733_00677, SAMEA3649758_02509, SAMEA3720909_04803, SAMEA3727630_01134, SAMEA3727643_04047, SAMEA3727679_02343, SAMEA3729663_00178, SAMEA4364603_01617, SAMEA4873619_01962, SAMEA4873632_01182
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): R3 Rosetta / References: UniProt: W9BAW3, AMP nucleosidase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Adenosine monophosphate nucleosidase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) R3 Rosetta
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
125 uMHEPES1
2500 mMsodium chlorideNaCl1
35 %glycerol1
42 mMDTT1
5.025 %sodium azide1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 96 % / Chamber temperature: 298 K / Details: 9 second blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 679
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
4cryoSPARC3.3.1CTF correction
7PHENIX1.18.2-3874model fitting
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC3.3.1classification
12cryoSPARC3.3.13D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1494578
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114781 / Num. of class averages: 17 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1T8R

1t8r


Accession code: 1T8R / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 82.81 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004521048
ELECTRON MICROSCOPYf_angle_d0.547528686
ELECTRON MICROSCOPYf_chiral_restr0.04443204
ELECTRON MICROSCOPYf_plane_restr0.00463702
ELECTRON MICROSCOPYf_dihedral_angle_d4.41452874
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints0.000705850679258
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000707263209159
ens_1d_4CELECTRON MICROSCOPYNCS constraints0.000713556213853
ens_1d_5CELECTRON MICROSCOPYNCS constraints0.000713736078961
ens_1d_6CELECTRON MICROSCOPYNCS constraints0.000715333618246

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