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- EMDB-12158: CryoEM structure of Mycobacterium tuberculosis UMP Kinase (UMPK) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12158
TitleCryoEM structure of Mycobacterium tuberculosis UMP Kinase (UMPK) in complex with UDP and UTP
Map data
Sample
  • Complex: UMPK-UDP-UTP
    • Protein or peptide: Uridylate kinase
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: URIDINE 5'-TRIPHOSPHATE
Function / homology
Function and homology information


UMP kinase / UMP kinase activity / 'de novo' CTP biosynthetic process / ATP binding / cytoplasm
Similarity search - Function
Uridylate kinase, bacteria / Uridylate kinase / Aspartate/glutamate/uridylate kinase / Amino acid kinase family / Acetylglutamate kinase-like superfamily
Similarity search - Domain/homology
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsBous J / Trapani S / Walter P / Bron P / Munier-Lehmann H
Funding supportEuropean Union, France, 2 items
OrganizationGrant numberCountry
Marie Sklodowska-Curie Actions, FragNET ITN675555European Union
Agence Nationale de la Recherche (ANR)ANR-10-INBS-05 France
CitationJournal: FEBS J / Year: 2022
Title: Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.
Authors: Patrick Walter / Ariel Mechaly / Julien Bous / Ahmed Haouz / Patrick England / Joséphine Lai-Kee-Him / Aurélie Ancelin / Sylviane Hoos / Bruno Baron / Stefano Trapani / Patrick Bron / ...Authors: Patrick Walter / Ariel Mechaly / Julien Bous / Ahmed Haouz / Patrick England / Joséphine Lai-Kee-Him / Aurélie Ancelin / Sylviane Hoos / Bruno Baron / Stefano Trapani / Patrick Bron / Gilles Labesse / Hélène Munier-Lehmann /
Abstract: Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate ...Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.
History
DepositionDec 24, 2020-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bes
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bes
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12158.png

Downloads & links

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Map

FileDownload / File: emd_12158.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 260 pix.
= 273.52 Å		1.05 Å/pix.
x 260 pix.
= 273.52 Å		1.05 Å/pix.
x 260 pix.
= 273.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.052 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0305 / Movie #1: 0.0305
Minimum - Maximum-0.119342916 - 0.22620551
Average (Standard dev.)1.4483821e-05 (±0.0057450947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 273.52002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0521.0521.052
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z273.520273.520273.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.1190.2260.000

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Supplemental data

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Mask #1

Fileemd_12158_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12158_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12158_half_map_2.map
Projections & Slices
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Sample components

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Entire : UMPK-UDP-UTP

EntireName: UMPK-UDP-UTP
Components
  • Complex: UMPK-UDP-UTP
    • Protein or peptide: Uridylate kinase
  • Ligand: URIDINE-5'-DIPHOSPHATE
  • Ligand: URIDINE 5'-TRIPHOSPHATE

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Supramolecule #1: UMPK-UDP-UTP

SupramoleculeName: UMPK-UDP-UTP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21/pDIA17
Molecular weightTheoretical: 177.558 KDa

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Macromolecule #1: Uridylate kinase

MacromoleculeName: Uridylate kinase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: UMP kinase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 29.625893 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MTEPDVAGAP ASKPEPASTG AASAAQLSGY SRVLLKLGGE MFGGGQVGLD PDVVAQVARQ IADVVRGGV QIAVVIGGGN FFRGAQLQQL GMERTRSDYM GMLGTVMNSL ALQDFLEKEG IVTRVQTAIT MGQVAEPYLP L RAVRHLEK ...String:
MGSSHHHHHH SSGLVPRGSH MTEPDVAGAP ASKPEPASTG AASAAQLSGY SRVLLKLGGE MFGGGQVGLD PDVVAQVARQ IADVVRGGV QIAVVIGGGN FFRGAQLQQL GMERTRSDYM GMLGTVMNSL ALQDFLEKEG IVTRVQTAIT MGQVAEPYLP L RAVRHLEK GRVVIFGAGM GLPYFSTDTT AAQRALEIGA DVVLMAKAVD GVFAEDPRVN PEAELLTAVS HREVLDRGLR VA DATAFSL CMDNGMPILV FNLLTDGNIA RAVRGEKIGT LVTT

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Macromolecule #2: URIDINE-5'-DIPHOSPHATE

MacromoleculeName: URIDINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: UDP
Molecular weightTheoretical: 404.161 Da
Chemical component information

ChemComp-UDP:
URIDINE-5'-DIPHOSPHATE / UDP*YM / Uridine diphosphate

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Macromolecule #3: URIDINE 5'-TRIPHOSPHATE

MacromoleculeName: URIDINE 5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: UTP
Molecular weightTheoretical: 484.141 Da
Chemical component information

ChemComp-UTP:
URIDINE 5'-TRIPHOSPHATE / UTP*YM / Uridine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
20.0 mMNa2HPO4
100.0 mMNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 53.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 793915
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 6 / Avg.num./class: 108000 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 88841
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Overall B value: 87.61
Output model

PDB-7bes:
CryoEM structure of Mycobacterium tuberculosis UMP Kinase (UMPK) in complex with UDP and UTP

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